Humanized anti-CD70 binding agents and uses thereof

ABSTRACT

Disclosed are CD70 binding agents, such as humanized anti-CD70 antibodies and fragments and derivatives, that exert a cytotoxic, cytostatic or immunomodulatory on CD70 expressing cells, as well as pharmaceutical compositions and kits comprising the antibody, fragment or derivative. Also disclosed are methods for the treatment of CD70-expressing cancers and immunological disorders, comprising administering to a subject the CD70 binding agents or pharmaceutical compositions.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/271,143,filed Oct. 11, 2011, now U.S. Pat. No. 8,562,987, which is acontinuation of U.S. application Ser. No. 11/912,096, filed Oct. 23,2008, now U.S. Pat. No. 8,067,546, which is a U.S. National StageApplication under 35 USC 371 of International Application No.PCT/US2006/015145, filed Apr. 19, 2006, which claims the benefit under35 USC 119(e) of U.S. Provisional Patent Application No. 60/673,070,filed Apr. 19, 2005, each of which is incorporated by reference hereinin its entirety.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“437966-Sequence.txt”, created on Oct. 14, 2013 and containing 52,029bytes, which is hereby incorporated by reference in its entirety for allpurposes.

BACKGROUND

CD70 is a member of the tumor necrosis factor (TNF) family of cellmembrane-bound and secreted molecules that are expressed by a variety ofnormal and malignant cell types. The primary amino acid (AA) sequence ofCD70 predicts a transmembrane type II protein with its carboxyl terminusexposed to the outside of cells and its amino terminus found in thecytosolic side of the plasma membrane (Bowman et al., 1994, J. Immunol.152:1756-61; Goodwin et al., 1993, Cell 73:447-56). Human CD70 iscomposed of a 20 AA cytoplasmic domain, an 18 AA transmembrane domain,and a 155 AA extracytoplasmic domain with two potential N-linkedglycosylation sites (Bowman et al., supra; Goodwin et al., supra).Specific immunoprecipitation of radioisotope-labeled CD70-expressingcells by anti-CD70 antibodies yields polypeptides of 29 and 50 kDa(Goodwin et al., supra; Hintzen et al., 1994, J. Immunol. 152:1762-73).Based on its homology to TNF-alpha and TNF-beta, especially instructural strands C, D, H and I, a trimeric structure is predicted forCD70 (Petsch et al., 1995, Mol. Immunol. 32:761-72).

Original immunohistological studies revealed that CD70 is expressed ongerminal center B cells and rare T cells in tonsils, skin, and gut(Hintzen et al., 1994, Int. Immunol. 6:477-80). Subsequently, CD70 wasreported to be expressed on the cell surface of recentlyantigen-activated T and B lymphocytes, and its expression wanes afterthe removal of antigenic stimulation (Lens et al., 1996, Eur. J.Immunol. 26:2964-71; Lens et al., 1997, Immunology 90:38-45). Within thelymphoid system, activated natural killer cells (Orengo et al., 1997,Clin. Exp. Immunol. 107:608-13) and mouse mature peripheral dendriticcells (Akiba et al., 2000, J. Exp. Med. 191:375-80) also express CD70.In non-lymphoid lineages, CD70 has been detected on thymic medullarepithelial cells (Hintzen et al., 1994, supra; Hishima et al., 2000, Am.J. Surg. Pathol. 24:742-46).

CD70 is not expressed on normal non-hematopoietic cells. CD70 expressionis mostly restricted to recently antigen-activated T and B cells underphysiological conditions, and its expression is down-regulated whenantigenic stimulation ceases. Evidence from animal models suggests thatCD70 may contribute to immunological disorders such as, e.g., rheumatoidarthritis (Brugnoni et al., 1997, Immunol. Lett. 55:99-104), psoriaticarthritis (Brugnoni et al., 1997, Immunol. Lett. 55:99-104), and lupus(Oelke et al., 2004, Arthritis Rheum. 50:1850-60). In addition to itspotential role in inflammatory responses, CD70 is also expressed on avariety of transformed cells including lymphoma B cells, Hodgkin's andReed-Sternberg cells, malignant cells of neural origin, and a number ofcarcinomas.

Accordingly, there is a need for anti-CD70 antibodies and other CD70binding agents that can exert a clinically useful cytotoxic, cytostatic,or immunomodulatory effect on CD70-expressing cells, particularlywithout exerting undesirable effects on non-CD70-expressing cells. Suchcompounds would be useful therapeutic agents against cancers thatexpress CD70 or immune disorders that are mediated by CD70-expressingcells. (The recitation of any reference in this application is not anadmission that the reference is prior art to this application.)

BRIEF SUMMARY

The present invention provides CD70 antibodies and related CD70 bindingagents and methods relating to the use of such binding agents for theprophylaxis or treatment of CD70-expressing cancers and immunologicaldisorders where CD70-expressing cells are present. The CD70 bindingagent, alone or in combination with a therapeutic agent, exerts acytotoxic, cytostatic, and/or immunomodulatory effect on CD70-expressingcells.

In an aspect, CD70 binding agents are provided. The CD70 binding agentcan be, for example, an antibody. In some embodiments, the binding agentincludes at least one effector domain mediating at least an ADCC, ADCPor CDC response in the subject. In some embodiments, the binding agentexerts a cytostatic, cytotoxic or immunomodulatory effect in the absenceof conjugation to a therapeutic agent. In some embodiments, the bindingagent is conjugated to a therapeutic agent that exerts a cytotoxic,cytostatic or immunodulatory effect. The antibody can compete forbinding to CD70 with monoclonal antibody 1F6 or 2F2.

In another aspect, a method of treating a CD70-expressing cancer in asubject is provided. The method generally includes administering to thesubject an effective amount of a CD70 binding agent. In someembodiments, the binding agent includes at least one effector domainmediating at least an ADCC, ADCP or CDC response in the subject. In someembodiments, the binding agent exerts a cytostatic, cytotoxic orimmunomodulatory effect in the absence of conjugation to a therapeuticagent. In some embodiments, the binding agent is conjugated to atherapeutic agent that exerts a cytotoxic, cytostatic or immunodulatoryeffect.

The CD70-binding agent can be, for example, an antibody. The antibodycan include, for example, an effector domain of a human IgM or IgGantibody. The IgG antibody can be, for example, a human IgG1 or IgG3subtype. In some embodiments, the antibody includes a human constantregion. In some embodiments, the CD70 binding agent competes for bindingto CD70 with monoclonal antibody 1F6 or 2F2. In other embodiments, theantibody is a humanized 1F6. In other embodiments, the antibody is ahumanized 2F2. The antibody can be, for example, monovalent, divalent ormultivalent.

The CD70-expressing cancer can be, a kidney tumor, a B cell lymphoma, acolon carcinoma, Hodgkin's Disease, multiple myeloma, Waldenström'smacroglobulinemia, non-Hodgkin's lymphoma, a mantle cell lymphoma,chronic lymphocytic leukemia, acute lymphocytic leukemia, anasopharyngeal carcinoma, brain tumor or a thymic carcinoma. The kidneytumor can be, for example, a renal cell carcinoma. The brain tumor canbe, for example, a glioma, a glioblastoma, an astrocytoma or ameningioma. The subject can be, for example, a mammal, such as a humanbeing.

In another aspect, a method for treating an immunological disorder isprovided. The method includes administering to a subject an effectiveamount of a CD70 binding agent. In some embodiments, the binding agentincludes at least one effector domain mediating at least an ADCC, ADCPor CDC response in the subject. In some embodiments, the binding agentexerts a cytostatic, cytotoxic or immunomodulatory effect in the absenceof conjugation to a therapeutic agent. In some embodiments, the bindingagent is conjugated to a therapeutic agent that exerts a cytotoxic,cytostatic or immunodulatory effect. The CD70 binding agent can be, forexample, an antibody. The antibody can include, for example, an effectordomain of a human IgM or IgG antibody. The IgG antibody can be, forexample, a human IgG₁ or IgG₃ subtype. In some embodiments, the antibodyincludes a human constant region.

The immunological disorder can be, for example, a T cell-mediatedimmunological disorder. In some embodiments, the T cell mediatedimmunological disorder comprises activated T cells expressing CD70. Insome embodiments, resting T cells are not substantially depleted byadministration of the antibody-drug conjugate. The T cell-mediatedimmunological disorder also can be, for example, rheumatoid arthritis,psoriatic arthritis, systemic lupus erythematosus (SLE), Type Idiabetes, asthma, atopic dermatitis, allergic rhinitis, thrombocytopenicpurpura, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto'sthyroiditis, Graves' disease, primary biliary cirrhosis, Wegener'sgranulomatosis, tuberculosis, or graft versus host disease. In otherembodiments, the immunological disorder is an activated B-lymphocytedisorder. The subject can be, for example, a mammal, such as a humanbeing.

In a related aspect, also provided is a pharmaceutical composition forthe treatment of a CD70-expressing cancer or an immunological disorder.The composition includes a CD70 binding agent and at least onepharmaceutically compatible ingredient. Further provided is apharmaceutical kit including a container including a CD70 binding agent,wherein the agent is lyophilized, and a second container comprising apharmaceutically acceptable diluent.

The present invention may be more fully understood by reference to thefollowing detailed description of the invention, non-limiting examplesof specific embodiments of the invention and the appended figures andsequence listing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of humanized 1F6 V_(H) humanized variants hV_(H)E(SEQ ID NO:6) and hV_(H)J (SEQ ID NO:14) with 1F6 mV_(H) (residues20-137 of SEQ ID NO:2) and human germline V_(H) exon V_(H)1-2 and J_(H)exon JH6 (collectively SEQ ID NO:31). In the alignment, a <•> indicatesthat the amino acid is identical to the murine residue. The highlightedlysine residue (K) at H46 of hV_(H)J indicates a back mutation to themurine residue. Underlined amino acid residues indicate positions inCDR1 and CDR2 according to the Kabat definition, whereas boxed residuesindicate positions in the corresponding CDR identified by the Chothiadefinition. A <^>, as at positions 37, 39, 45, 47, 95 and 97, indicatesa residue in involved in the V_(H)/V_(L) interface.

FIG. 2 is an alignment of humanized 1F6 V_(H) humanized variants hV_(H)H(SEQ ID NO:10) and hV_(H)M (SEQ ID NO:18) with 1F6 mV_(H) (residues20-137 of SEQ ID NO:2) and human germline V_(H) exon V_(H)1-18 and J_(H)exon JH6 (collectively SEQ ID NO:32). In the alignment, a <•> indicatesthat the amino acid is identical to the murine residue. The highlightedresidues at H46, H67, H68, H69, H70, and H71 of hV_(H)M indicate backmutations to the murine residues. Similarly, the highlighted residues atH67, H68, H69, H70, H71, H80, H81, H82, and H82A of hV_(H)H indicateback mutations to the murine residues. Underlined amino acid residuesindicate positions in CDR1, CDR2, and CDR3 according to the Kabatdefinition, whereas boxed residues indicate positions in thecorresponding CDR identified by the Chothia definition. A <^>, as atpositions 37, 39, 45, 47, 98, and 100 indicates a residue in involved inthe V_(H)/V_(L) interface.

FIG. 3 is an alignment of humanized 1F6 V_(L) variant hV_(L)A (SEQ IDNO:24) with 1F6 mV_(L) (residues 21-132 of SEQ ID NO:22) and humangermline V_(κ) exon B3 and J_(κ) exon Jκ-1 (collectively SEQ ID NO:33).In the alignment, a <•> indicates that the amino acid is identical tothe murine residue. Underlined amino acid residues indicate positions inCDR1, CDR2, and CDR3 according to the Kabat definition, whereas boxedresidues indicate positions in the corresponding CDR identified by theChothia definition. A <^>, as at positions 42, 44, 50, 52, and 93,indicates a residue in involved in the V_(H)/V_(L) interface.

FIGS. 4A and 4B show that humanized 1F6 anti-CD70 antibodies mediateantibody-dependent cellular cytotoxicity (ADCC). Na₂ ⁵¹CrO₄-labeledtarget cells (WIL2-S B lymphoblastoid cells, 786-O renal cell carcinomacells, and 769-P renal cell carcinoma cells) were coated with antibodyand incubated with peripheral blood mononuclear cells (PBMC) at aneffector to target ratio of 10 CD16⁺ (FcγIII receptor) cells to 1 targetcell. After 4 hours, the supernatants from lysed cells were measured ona scintillation counter. The percent specific lysis was calculated as{(test sample cpm−spontaneous cpm)±(total cpm−spontaneous cpm)}×100.Points represent the mean±standard deviation of triplicate samples. FIG.4A shows the ADCC activity mediated by humanized 1F6 variants HHLA, HJLAand HELA compared to non-binding antibody control hIgG₁ and murine 1F6antibody. FIG. 4B shows antibody-directed lysis of renal cell carcinomacell lines mediated by chimeric 1F6 and humanized 1F6 variant HJLA, andhIgG₁.

FIG. 5 shows that humanized 1F6 anti-CD70 variant HJLA mediatescomplement-dependent cellular cytotoxicity (CDC). LP-1, MHH PreB-1 andWIL-S target cells were mixed with chimeric 1F6, humanized 1F6 (HJLA) ornon-binding human Ig in the presence of human serum as a source ofcomplement. After 2 hours at 37° C., propidium iodide was added todetermine cell viability as measured by flow cytometry, and the amountof lytic activity was calculated. Bars represent the mean±standarddeviation of triplicate samples.

FIG. 6 shows that humanized 1F6 anti-CD70 antibodies mediateantibody-dependent cellular phagocytosis (ADCP). 786-O CD70⁺ renal cellcarcinoma target cells were labeled with a red fluorescent cell membranedye (PKH26, Sigma-Aldrich, Inc., St. Louis, Mo.) and then coated withchimeric 1F6, humanized 1F6 (HJLA) or non-binding human Ig for 30minutes on ice. Labeled, antibody-treated target cells were mixed withmonocyte-derived macrophages at a ratio of 1 macrophage to 4 targetcells for 1 hour at 37° C. Macrophages were stained with an Alexa Fluor®488 (Molecular Probes, Inc., Eugene, Oreg.) anti-CD11b antibody and thepercent phagocytic activity was determined by the percentage ofmacrophages exhibiting dual fluorescence when analyzed by flowcytometry.

FIG. 7 shows humanized 1F6 anti-CD70 antibody prolongs survival of micein xenograft models of disseminated lymphoma and multiple myeloma. (A)Survival of mice injected with Raji cells and treated with humanized 1F6antibody or control non-binding antibody. Treatment was initiated oneday after tumor cell injection and was administered by intraperitonealinjection every four days for a total of six doses (n=10 per group). (B,C, left panels) Survival of mice injected with L363- or MM.1S-cells andtreated with humanized 1F6 starting one day after cell implant. Theantibody was administered by intravenous injection once weekly for atotal of five doses. Mice were monitored twice weekly and wereeuthanized upon manifestation of disease (n=7 per group). (B, C, rightpanels) Analysis of λ light chain concentrations in sera collected frommice injected with L363- or MM.1S-cells. Samples were collected on days35 and 42 post tumor injection, respectively. In all studies, p valuesgiven are between humanized 1F6-treated groups and the untreated group.

FIG. 8 shows humanized 1F6 mediates depletion of antigen-specificCD8+/Vβ17+ cells. PBMCs from a normal HLA-A0201 donor were stimulatedwith the M1 peptide. (A) Peptide-stimulated cultures were untreated ortreated with concurrent addition of graded doses of humanized 1F6antibody, as indicated. The percent CD8+/Vβ17+ cells after 9 days wasdetermined by flow cytometry. (B) Peptide-stimulated cultures wereuntreated or treated on day 0 with 1 μg/ml humanized 1F6 in the absence(solid bars) or presence (hatched bars) of 10 μg/ml antibody specificfor FcγRIII (CD16). The percent CD8+/Vβ7+ cells after 9 days wasdetermined by flow cytometry.

FIG. 9 shows minimal impact of anti-CD70 1F6 antibody on bystanderresting T cells. PBMCs from a normal HLA-A0201 donor were untreated (nostim) or stimulated with M1 peptide (peptide stim) in the presence orabsence of 1 μg/mL c1F6. After nine days in culture, Vβ TCRrepresentation among CD4 and CD8 cells from each group was analyzed byflow cytometry using the IOTest® Beta Mark TCR Vβ Repertoire Kit.

FIG. 10 shows a mouse Xenograft model of Renal Cell Carcinoma. (A)Subcutaneous 786-O tumors were initiated in nude mice by implantingtumor fragments (N=5 or 6/group) of approximately 30 mm³. Tumor growthwas allowed to establish and treatment began when average tumor sizewithin each group was approximately 100 mm³ h1F6-mcMMAF4 or h1F6-vcMMAF4at the indicated doses was administered at a q4d×4 schedule beginning onday 17 after tumor implantation, as indicated by the arrows.Cross-strikes indicate when animals with tumors >1000 mm³ wereeuthanized. (B) 786-O tumor implantation and treatment initiation arethe same as given in (A). Groups of mice (N=5-7) were administered withh1F6-mcMMAF4 or h1F6-vcMMAF4 at 0.17 mg/kg at a q4d×4 or q4d×10 schedulebeginning on day 13 after tumor implantation. Tumor growth isrepresented by Kaplan-Meier plots. An event was registered when a mousewith a tumor quadrupled in size compared to day 13 when treatment began.Mice with tumors that did not quadruple in size at the end of theexperiment on day 43 were censored. The log-rank test was used togenerate p values between treatment groups and the untreated group.

FIG. 11 shows a mouse Xenograft Model of Multiple Myeloma. (A) Tenmillion MM-1S cells were injected intravenously into each SCID mouse.Groups of mice (N=8-10) were left untreated, received IgG-vcMMAF4,IgG-mcMMAF4, h1F6-vcMMAF4, or h1F6-mcMMAF4 at the specified doses on aq7d×5 schedule, as indicated by the arrows. Mice showing symptoms ofhind limb paralysis, hunched posture, cranial swelling, and/or scruffycoat were euthanized, and the percent survival of each group wasplotted. The log-rank test was used to generate p values betweentreatment groups and the control groups. (B) Bone marrow cells wererecovered from the femurs of euthanized mice due to the above diseasesymptoms or on day 122 post tumor cell implantation when the experimentwas terminated. The percentage of CD138-expressing MM-1S cells in thefemers of each mouse was determined by flow cytometry. The Mann-Whitneytest was used to derived p values between the indicated groups.

FIG. 12 shows a mouse Xenograft model of Multiple Myeloma. (A) Tenmillion L363 cells were injected intravenously into each SCID mouse.Groups of mice (N=7) were left untreated, received IgG-vcMMAF4, orh1F6-vcMMAF4 at the specified doses on a q7d×5 schedule as indicated bythe arrows. Mice showing palpable tumor masses were euthanized, and thepercent survival of each group was plotted. The log-rank test was usedto generate the p value between the treated group and the untreatedgroup. (B) Serum samples were obtained from mice 40 days after tumorimplant. The concentration of human λ light chain in the serum of eachmouse was determined by ELISA. The Mann-Whitney test was used to derivep values between the indicated groups.

DETAILED DESCRIPTION

The present invention provides CD70 binding agents and methods for usingsuch binding agents for the prophylaxis or treatment of CD70-expressingcancers and immunological disorders. The CD70 binding agent specificallybinds to CD70 (e.g., the extracellular domain). The binding agent mayinclude at least one effector domain mediating an ADCC, ADCP and/or CDCresponse. The binding agent may exert a cytostatic, cytotoxic orimmunomodulatory effect in the absence of conjugation to a therapeuticagent. The binding agent may be conjugated to a therapeutic agent thatexerts a cytotoxic, cytostatic or immunodulatory effect.

In one aspect, the compositions and methods relate to CD70 bindingagents, such as antibodies and antibody derivatives. The anti-CD70antibody can be a monoclonal, chimeric or humanized antibody, or afragment or derivative thereof. In some embodiments, the anti-CD70antibody includes an antibody constant region or domain. The antibodyconstant region or domain can be, for example, of the IgG subtype. In anexemplary embodiment, the anti-CD70 antibody, fragment or derivativesthereof, competes with the murine monoclonal antibody (mAb) 1F6 or 2F2for binding to CD70 and comprises human antibody constant regionsequences. In another exemplary embodiment, the anti-CD70 antibody, orfragment or derivative thereof, has an effector domain (e.g., an Fcportion) that can interact with effector cells or complement to mediatea cytotoxic, cytostatic, and/or immunomodulatory effect that results inthe depletion or inhibition of the proliferation of CD70-expressingcells. In another exemplary embodiment, the anti-CD70 antibody lackseffector function. In another exemplary embodiment, the anti-CD70antibody is conjugated to a therapeutic agent.

Also included are kits and articles of manufacture comprising a CD70binding agent (e.g., a humanized anti-CD70 antibody).

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

I. DEFINITIONS AND ABBREVIATIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. When tradenames are used herein, applicants intend to independently include thetrade name product formulation, the generic drug, and the activepharmaceutical ingredient(s) of the trade name product. As used herein,the following terms and phrases have the meanings ascribed to themunless specified otherwise.

The terms “CD70 binding agent” and “anti-CD70 binding agent” as usedherein means an anti-CD70 antibody, a derivative or a fragment of ananti-CD70 antibody, or other agent that binds to CD70 and comprises atleast one CDR or variable region of a CD70 binding antibody, or aderivative thereof.

The term “specifically binds” means that the binding agent will react,in a highly selective manner, with its corresponding antigen and notwith the multitude of other antigens (e.g., non-CD70 molecules).

As used herein, the term “functional” in the context of a CD70 bindingagent indicates that the binding agent is capable of binding to CD70.

The terms “inhibit” or “inhibition of” as used herein means to reduce bya measurable amount, or to prevent entirely.

The term “deplete” in the context of the effect of a CD70-binding agenton CD70-expressing cells refers to a reduction in the number of orelimination of the CD70-expressing cells.

“Intact antibodies” and “intact immunoglobulins” are defined herein asheterotetrameric glycoproteins, typically of about 150,000 daltons,composed of two identical light (L) chain and two identical heavy (H)chains. Each light chain is covalently linked to a heavy chain by adisulfide bond to form a heterodimer. The heterotetramer is formed bycovalent disulfide linkage between the two identical heavy chains ofsuch heterodimers. Although the light and heavy chains are linkedtogether by a disulfide bond, the number of disulfide linkages betweenthe two heavy chains varies by immunoglobulin (Ig) isotype. Each heavyand light chain also has regularly spaced intrachain disulfide bridges.Each heavy chain has at the amino-terminus a variable domain (V_(H)),followed by three or four constant domains (C_(H)1, C_(H)2, C_(H)3,and/or C_(H)4), as well as a hinge (J) region between C_(H)1 and C_(H)2.Each light chain has two domains, an amino-terminal variable domain(V_(L)) and a carboxy-terminal constant domain (C_(L)). The V_(L) domainassociates non-covalently with the V_(H) domain, whereas the C_(L)domain is commonly covalently linked to the C_(H)1 domain via adisulfide bond. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains (Chothia etal., 1985, J. Mol. Biol. 186:651-663).

The term “hypervariable” refers to certain sequences within the variabledomains that differ extensively in sequence among antibodies and containresidues that are directly involved in the binding and specificity ofeach particular antibody for its specific antigenic determinant.Hypervariability, both in the light chain and the heavy chain variabledomains, is concentrated in three segments known as complementaritydetermining regions (CDRs) or hypervariable loops (HVLs). CDRs aredefined by sequence comparison in Kabat et al., 1991, In: Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, M.D., whereas HVLs arestructurally defined according to the three-dimensional structure of thevariable domain, as described by Chothia and Lesk, 1987, J. Mol. Biol.196:901-917. Where these two methods result in slightly differentidentifications of a CDR, the structural definition is preferred. Asdefined by Kabat (see Kabat et al., “Sequences of proteins ofimmunological interest, 5th ed., Pub. No. 91-3242, U.S. Dept. Health &Human Services, NIH, Bethesda, M.D., 1991), CDR-L1 is positioned atabout residues 24-34, CDR-L2, at about residues 50-56, and CDR-L3, atabout residues and 89-97 in the light chain variable domain and at about31-35 in CDR-H1, at about 50-65 in CDR-H2, and at about 95-102 in CDR-H3in the heavy chain variable domain.

The three CDRs within each of the heavy and light chains are separatedby framework regions (FRs), which contain sequences that tend to be lessvariable. From the amino terminus to the carboxy terminus of the heavyand light chain variable domains, the FRs and CDRs are arranged in theorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The largely β-sheetconfiguration of the FRs brings the CDRs within each of the chains toclose proximity to each other as well as to the CDRs from the otherchain. The resulting conformation contributes to the antigen bindingsite (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages647-669), although not all CDR residues are necessarily directlyinvolved in antigen binding.

FR residues and Ig constant domains typically are not directly involvedin antigen binding, but can contribute to antigen binding or mediateantibody effector function. Some FR residues can have a significanteffect on antigen binding in at least three ways: by noncovalentlybinding directly to an epitope, by interacting with one or more CDRresidues, and by affecting the interface between the heavy and lightchains. The constant domains mediate various Ig effector functions, suchas participation of the antibody in antibody dependent cellularcytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and/orantibody dependent cellular phagocytosis (ADCP).

The light chains of vertebrate immunoglobulins are assigned to one oftwo clearly distinct classes, kappa (k) and lambda (λ), based on theamino acid sequence of the constant domain. By comparison, the heavychains of mammalian immunoglobulins are assigned to one of five majorclasses, according to the sequence of the constant domains: IgA, IgD,IgE, IgG, and IgM. IgG and IgA are further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of the classes of nativeimmunoglobulins are well known.

The terms “antibody”, “anti-CD70 antibody”, “humanized anti-CD70antibody”, and “variant humanized anti-CD70 antibody” are used herein inthe broadest sense and specifically encompass full-length and nativeantibodies, monoclonal antibodies (including full-length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody or antigen-binding fragmentsthereof, such as variable domains and other portions of antibodies thatexhibit a desired biological activity, e.g., CD70 binding.

The term “monoclonal antibody” (mAb) refers to an antibody obtained froma population of substantially homogeneous antibodies; that is, theindividual antibodies comprising the population are identical except fornaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic determinant, also referred to as an epitope. Themodifier “monoclonal” is indicative of a substantially homogeneouspopulation of antibodies directed to the identical epitope and is not tobe construed as requiring production of the antibody by any particularmethod. Monoclonal antibodies can be made by any technique ormethodology known in the art; for example, the hybridoma method firstdescribed by Köhler et al., 1975, Nature 256:495, or recombinant DNAmethods known in the art (see, e.g., U.S. Pat. No. 4,816,567). Inanother example, monoclonal antibodies can also be isolated from phageantibody libraries, using techniques described in Clackson et al., 1991,Nature 352: 624-628, and Marks et al., 1991, J. Mol. Biol. 222:581-597.

In contrast, the antibodies in a preparation of polyclonal antibodiesare typically a heterogeneous population of immunoglobulin isotypesand/or classes and also exhibit a variety of epitope specificity.

The term “chimeric” antibody, as used herein, is a type of monoclonalantibody in which a portion of or the complete amino acid sequence inone or more regions or domains of the heavy and/or light chain isidentical with, homologous to, or a variant of the correspondingsequence in a monoclonal antibody from another species or belonging toanother immunoglobulin class or isotype, or from a consensus sequence.Chimeric antibodies include fragments of such antibodies, provided thatthe antibody fragment exhibits the desired biological activity of itsparent antibody, for example binding to the same epitope (see, e.g.,U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad.Sci. USA 81:6851-6855). Methods for producing chimeric antibodies areknown in the art. (See, e.g., Morrison, 1985. Science 229:1202; Oi etal., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol.Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and4,816,397.)

The terms “antibody fragment”, “anti-CD70 antibody fragment”, “humanizedanti-CD70 antibody fragment”, and “variant humanized anti-CD70 antibodyfragment” refer to a portion of a full-length anti-CD70 antibody inwhich a variable region or a functional capability is retained, forexample, specific CD70 epitope binding. Examples of antibody fragmentsinclude, but are not limited to, a Fab, Fab′, F(ab′)₂, Fd, Fv, scFv andscFv-Fc fragment, diabody, triabody, tetrabody, linear antibody,single-chain antibody, and other multispecific antibodies formed fromantibody fragments. (See Holliger and Hudson, 2005, Nat. Biotechnol.23:1126-1136.)

A “single-chain Fv” or “scFv” antibody fragment is a single chain Fvvariant comprising the V_(H) and V_(L) domains of an antibody, in whichthe domains are present in a single polypeptide chain and which iscapable of recognizing and binding antigen. The scFv polypeptideoptionally contains a polypeptide linker positioned between the V_(H)and V_(L) domains that enables the scFv to form a desiredthree-dimensional structure for antigen binding (see, e.g., Pluckthun,1994, In The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburgand Moore eds., Springer-Verlag, New York, pp. 269-315).

The term “diabody” refers to small antibody fragment having twoantigen-binding sites. Each fragment contains a heavy chain variabledomain (V_(H)) concatenated to a light chain variable domain (V_(L)) toform a V_(H)-V_(L) or V_(L)-V_(H) polypeptide. By using a linker that istoo short to allow pairing between the two domains on the same chain,the linked V_(H)-V_(L) domains are forced to pair with complementarydomains of another chain, creating two antigen-binding sites. Diabodiesare described more fully, for example, in EP 404 097; WO 93/11161; andHollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448.

The term “linear antibody” refers to antibodies that comprises a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) that form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific, as described in Zapata et al., 1995, Protein Eng.8(10):1057-1062.

A “humanized antibody” refers to an immunoglobulin amino acid sequencevariant or fragment thereof which is capable of binding to apredetermined antigen and which comprises a variable region polypeptidechain having framework regions having substantially the amino acidsequence of a human immunoglobulin and a CDR(s) having substantially theamino acid sequence of a non-human immunoglobulin.

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are referred to herein as “import” residues, whichare typically taken from an “import” antibody domain, particularly avariable domain. An import residue, sequence, or antibody has a desiredaffinity and/or specificity, or other desirable antibody biologicalactivity as discussed herein.

In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the framework regions arethose of a human immunoglobulin sequence, such as from, for example, aconsensus or germline sequence. The humanized antibody optionally alsowill comprise at least a portion of an immunoglobulin Fc domain,typically that of a human immunoglobulin. For example, the antibody maycontain both the light chain as well as at least the variable domain ofa heavy chain. The antibody also may include the C_(H)1, hinge (J),C_(H)2, C_(H)3, and/or C_(H)4 regions of the heavy chain, asappropriate.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG₁, IgG₂, IgG₃ and IgG₄. The constant region or domain caninclude, for example, a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity (e.g.,IgG₁). Where such cytotoxic activity is not desirable, the constantdomain may be of another class (e.g., IgG₂). The humanized antibody maycomprise sequences from more than one class or isotype, and selectingparticular constant domains to optimize desired effector functions iswithin the ordinary skill in the art.

The FR and CDR regions of the humanized antibody need not correspondprecisely to the parental sequences, e.g., the import CDR or theconsensus FR may be altered by substitution, insertion or deletion of atleast one residue so that the CDR or FR residue at that site does notcorrespond to either the consensus or the import antibody. Suchmutations typically will not be extensive. Usually, at least 75% of thehumanized antibody residues will correspond to those of the parental FRand CDR sequences, more often at least 90%, and most often greater than95%.

The term “antibody effector function(s)” as used herein refers to afunction contributed by an Fc domain(s) of an Ig. Such functions can be,for example, antibody-dependent cellular cytotoxicity,antibody-dependent cellular phagocytosis or complement-dependentcytotoxicity. Such function can be effected by, for example, binding ofan Fc effector domain(s) to an Fc receptor on an immune cell withphagocytic or lytic activity or by binding of an Fc effector domain(s)to components of the complement system. Typically, the effect(s)mediated by the Fc-binding cells or complement components result ininhibition and/or depletion of the CD70 targeted cell. Without intendingto be bound by any particular theory, Fc regions of antibodies canrecruit Fc receptor (FcR)-expressing cells and juxtapose them withantibody-coated target cells. Cells expressing surface FcR for IgGsincluding FcγRIII (CD16), FcγRII (CD32) and FcγRIII (CD64) can act aseffector cells for the destruction of IgG-coated cells. Such effectorcells include monocytes, macrophages, natural killer (NK) cells,neutrophils and eosinophils. Engagement of FcγR by IgG activatesantibody-dependent cellular cytotoxicity (ADCC) or antibody-dependentcellular phagocytosis (ADCP). ADCC is mediated by CD16⁺ effector cellsthrough the secretion of membrane pore-forming proteins and proteases,while phagocytosis is mediated by CD32⁺ and CD64⁺ effector cells (seeFundamental Immunology, 4^(th) ed., Paul ed., Lippincott-Raven, N.Y.,1997, Chapters 3, 17 and 30; Uchida et al., 2004, J. Exp. Med.199:1659-69; Akewanlop et al., 2001, Cancer Res. 61:4061-65; Watanabe etal., 1999, Breast Cancer Res. Treat. 53:199-207). In addition to ADCCand ADCP, Fc regions of cell-bound antibodies can also activate thecomplement classical pathway to elicit complement-dependent cytotoxicity(CDC). C1q of the complement system binds to the Fc regions ofantibodies when they are complexed with antigens. Binding of C1q tocell-bound antibodies can initiate a cascade of events involving theproteolytic activation of C4 and C2 to generate the C3 convertase.Cleavage of C3 to C3b by C3 convertase enables the activation ofterminal complement components including C5b, C6, C7, C8 and C9.Collectively, these proteins form membrane-attack complex pores on theantibody-coated cells. These pores disrupt the cell membrane integrity,killing the target cell (see Immunobiology, 6^(th) ed., Janeway et al.,Garland Science, N.Y., 2005, Chapter 2).

The term “antibody-dependent cellular cytotoxicity”, or ADCC, is amechanism for inducing cell death that depends upon the interaction ofantibody-coated target cells with immune cells possessing lytic activity(also referred to as effector cells). Such effector cells includenatural killer cells, monocytes/macrophages and neutrophils. Theeffector cells attach to an Fc effector domain(s) of Ig bound to targetcells via their antigen-combining sites. Death of the antibody-coatedtarget cell occurs as a result of effector cell activity.

The term “antibody-dependent cellular phagocytosis”, or ADCP, refers tothe process by which antibody-coated cells are internalized, either inwhole or in part, by phagocytic immune cells (e.g., macrophages,neutrophils and dendritic cells) that bind to an Fc effector domain(s)of Ig.

The term “complement-dependent cytotoxicity”, or CDC, refers to amechanism for inducing cell death in which an Fc effector domain(s) of atarget-bound antibody activates a series of enzymatic reactionsculminating in the formation of holes in the target cell membrane.Typically, antigen-antibody complexes such as those on antibody-coatedtarget cells bind and activate complement component C1q which in turnactivates the complement cascade leading to target cell death.Activation of complement may also result in deposition of complementcomponents on the target cell surface that facilitate ADCC by bindingcomplement receptors (e.g., CR3) on leukocytes.

“Immune cell” as used herein refers to a cell of hematopoietic lineageinvolved in regulating an immune response. In typical embodiments, animmune cell is a T lymphocyte, a B lymphocyte, an NK cell, amonocyte/macrophage, or a dendritic cell.

“Effector cell” as used herein refers to a cell that expresses a surfacereceptor for the Fc domain of an immunoglobulin (FcR). For example,cells that express surface FcR for IgGs including FcγRIII (CD16), FcγRII(CD32) and FcγRIII (CD64) can act as effector cells. Such effector cellsinclude monocytes, macrophages, natural killer (NK) cells, neutrophilsand eosinophils.

A “therapeutic agent” is an agent that exerts a cytotoxic, cytostatic,and/or immunomodulatory effect on cancer cells, activated immune cellsor other target cell population. Examples of therapeutic agents includecytotoxic agents, chemotherapeutic agents, cytostatic agents, andimmunomodulatory agents.

A “cytotoxic effect” refers to the depletion, elimination and/or thekilling of a target cell. A “cytotoxic agent” refers to an agent thathas a cytotoxic effect on a cell. The term is intended to includeradioactive isotopes (such as I¹³¹, I¹²⁵, Y⁹⁰, and Re¹⁸⁶),chemotherapeutic agents, and toxins such as enzymatically active toxinsof bacterial, fungal, plant, or animal origin, and fragments thereof.Such cytotoxic agents can be coupled to an antibody, e.g., a humanizedanti-CD70 antibody, and used, for example, to treat a patient indicatedfor therapy with the antibody. In one embodiment, “cytotoxic agent”includes monoclonal antibodies, e.g., antibodies used in combinationwith the humanized antibodies described herein.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such a thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan, and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin, and bizelesin syntheticanalogues) and derivatives thereof; cryptophycines (particularlycryptophycin 1 and cryptophycin 8); dolastatin, auristatins (includinganalogues monomethyl-auristatin E and monomethyl-auristatin F (see,e.g., U.S. Published Application No. 2005-0238649, published Oct. 27,2005, incorporated herein in its entirety); duocarmycin (including thesynthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin;sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,chlornaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine; trofosfamide, uracil mustard;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine; antibiotics such as the enediyne antibiotics(e.g., calicheamicin, especially calichemicin gamma1I and calicheamicinphiI1, see for example, Agnew, Chem. Intl. Ed. Engl., 33:183-186;dynemicin, including dynemicin A; bisphosphonates, such as clodronate;esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(Adriamycin™) (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, anddeoxydoxorubicin), epirubucin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycine, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such a methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adranals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; democolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone, mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitabronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (Navelbine™); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids, or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including Nolvadex™), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston™); aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (Megace™), exemestane, formestane, fadrozole, vorozole(Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; and pharmaceutically acceptable salts, acids, orderivatives of any of the above.

The term “prodrug” as used herein refers to a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.See, for example, Wilman, 1986, “Prodrugs in Cancer Chemotherapy”, InBiochemical Society Transactions, 14, pp. 375-382, 615th MeetingBelfast; and Stella et al., 1985, “Prodrugs: A Chemical Approach toTargeted Drug Delivery, In: “Directed Drug Delivery, Borchardt et al.,(ed.), pp. 247-267, Humana Press. Useful prodrugs include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs, and optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs that can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form include, but are not limited to, thosechemotherapeutic agents described above.

A “cytostatic effect” refers to the inhibition of cell proliferation. A“cytostatic agent” refers to an agent that has a cytostatic effect on acell, thereby inhibiting the growth and/or expansion of a specificsubset of cells.

The term “immunomodulatory effect” as used herein refers to astimulation (immunostimulatory) or inhibition (immunosuppressive) of thedevelopment or maintenance of an immunologic response Inhibition can beeffected by, for example, by elimination of immune cells (e.g., T or Blymphocytes); induction or generation of immune cells that can modulate(e.g., down-regulate) the functional capacity of other cells; inductionof an unresponsive state in immune cells (e.g., anergy); or increasing,decreasing or changing the activity or function of immune cells,including, for example, altering the pattern of proteins expressed bythese cells (e.g., altered production and/or secretion of certainclasses of molecules such as cytokines, chemokines, growth factors,transcription factors, kinases, costimulatory molecules or other cellsurface receptors, and the like). An “immunomodulatory agent” refers toan agent that has an immunomodulatory effect on a cell. In someembodiments, an immunomodulatory agent has a cytotoxic or cytostaticeffect on an immune cell that promotes an immune response.

The term “label” refers to a detectable compound or composition that isconjugated directly or indirectly to the antibody. The label may itselfbe detectable (e.g., radioisotope labels or fluorescent labels) or, inthe case of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition that is detectable. Labeled anti-CD70antibody can be prepared and used in various applications including invitro and in vivo diagnostics.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordinarilyexpress the antibody where, for example, the nucleic acid molecule is ina chromosomal location different from that of natural cells.

The term “control sequences” refers to polynucleotide sequencesnecessary for expression of an operably linked coding sequence in aparticular host organism. The control sequences suitable for use inprokaryotic cells include, for example, promoter, operator, and ribosomebinding site sequences. Eukaryotic control sequences include, but arenot limited to, promoters, polyadenylation signals, and enhancers. Thesecontrol sequences can be utilized for expression and production ofanti-CD70 binding agent in prokaryotic and eukaryotic host cells.

A nucleic acid sequence is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,a nucleic acid presequence or secretory leader is operably linked to anucleic acid encoding a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide; a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and, in the case of a secretory leader,contiguous and in reading frame. However, enhancers are optionallycontiguous. Linking can be accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers can be used to link the DNA sequences.

The term “polypeptide” refers to a polymer of amino acids and itsequivalent and does not refer to a specific length of a product; thus,“peptides” and “proteins” are included within the definition of apolypeptide. Also included within the definition of polypeptides are“antibodies” as defined herein. A “polypeptide region” refers to asegment of a polypeptide, which segment may contain, for example, one ormore domains or motifs (e.g., a polypeptide region of an antibody cancontain, for example, one or more complementarity determining regions(CDRs)). The term “fragment” refers to a portion of a polypeptidetypically having at least 20 contiguous or at least 50 contiguous aminoacids of the polypeptide. A “derivative” is a polypeptide or fragmentthereof having one or more non-conservative or conservative amino acidsubstitutions relative to a second polypeptide; or a polypeptide orfragment thereof that is modified by covalent attachment of a secondmolecule such as, e.g., by attachment of a heterologous polypeptide, orby glycosylation, acetylation, phosphorylation, and the like. Furtherincluded within the definition of “derivative” are, for example,polypeptides containing one or more analogs of an amino acid (e.g.,unnatural amino acids and the like), polypeptides with unsubstitutedlinkages, as well as other modifications known in the art, bothnaturally and non-naturally occurring.

An “isolated” polypeptide is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. An isolated polypeptide includes an isolatedantibody, or a fragment or derivative thereof. “Antibody” includes theantibody in situ within recombinant cells since at least one componentof the antibody's natural environment will not be present.

In certain embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, and inother aspects to more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain.

The term “heterologous,” in the context of a polypeptide, means from adifferent source (e.g., a cell, tissue, organism, or species) ascompared with another polypeptide, so that the two polypeptides aredifferent. Typically, a heterologous polypeptide is from a differentspecies.

In the context of immunoglobulin polypeptides or fragments thereof,“conservative substitution” means one or more amino acid substitutionsthat do not substantially reduce specific binding (e.g., as measured bythe K_(D)) of the immunoglobulin polypeptide or fragment thereof to anantigen (i.e., substitutions that increase binding affinity, that do notsignificantly alter binding affinity, or that reduce binding affinity byno more than about 40%, typically no more than about 30%, more typicallyno more than about 20%, even more typically no more than about 10%, ormost typically no more than about 5%, as determined by standard bindingassays such as, e.g., ELISA).

The terms “identical” or “percent identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence. To determine thepercent identity, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In some embodiments, the two sequences are the samelength.

The term “substantially identical,” in the context of two nucleic acidsor polypeptides, refers to two or more sequences or subsequences thathave at least 50%, at least 55%, at least 60%, or at least 65% identity;typically at least 70% or at least 75% identity; more typically at least80% or at least 85% identity; and even more typically at least 90%, atleast 95%, or at least 98% identity (e.g., as determined using one ofthe methods set forth infra).

The terms “similarity” or “percent similarity” in the context of two ormore polypeptide sequences refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are the same or conservatively substituted when compared andaligned for maximum correspondence, as measured using one of the methodsset forth infra. By way of example, a first amino acid sequence can beconsidered similar to a second amino acid sequence when the first aminoacid sequence is at least 50%, 60%, 70%, 75%, 80%, 90%, or 95%identical, or conservatively substituted, to the second amino acidsequence when compared to an equal number of amino acids as the numbercontained in the first sequence, or when compared to an alignment ofpolypeptides that has been aligned by, e.g., one of the methods setforth infra.

The terms “substantial similarity” or “substantially similar,” in thecontext of polypeptide sequences, indicate that a polypeptide region hasa sequence with at least 70%, typically at least 80%, more typically atleast 85%, or at least 90% or at least 95% sequence similarity to areference sequence. For example, a polypeptide is substantially similarto a second polypeptide, for example, where the two peptides differ byone or more conservative substitution(s).

In the context of anti-CD70 antibodies, or derivatives thereof, aprotein that has one or more polypeptide regions substantially identicalor substantially similar to one or more antigen-binding regions (e.g., aheavy or light chain variable region, or a heavy or light chain CDR) ofan anti-CD70 antibody retains specific binding to an epitope of CD70recognized by the anti-CD70 antibody, as determined using any of variousstandard immunoassays known in the art or as referred to herein.

The determination of percent identity or percent similarity between twosequences can be accomplished using a mathematical algorithm. Apreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of two sequences is the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleicacid encoding a protein of interest. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3, to obtainamino acid sequences homologous to protein of interest. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Anothernon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. Additional algorithms for sequenceanalysis are known in the art and include ADVANCE and ADAM as describedin Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTAdescribed in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA. Alternatively, protein sequence alignment may be carried outusing the CLUSTAL W algorithm, as described by Higgins et al., 1996,Methods Enzymol. 266:383-402.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include the progenythereof. Thus, “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or naturallyoccurring mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

The term “subject” for purposes of treatment refers to any animal,particularly an animal classified as a mammal, including humans,domesticated and farm animals, and zoo, sports, or pet animals, such asdogs, horses, cats, cows, and the like. Preferably, the subject ishuman.

A “disorder”, as used herein, and the terms “CD70-associated disorder”and “CD70-associated disease” refer to any condition that would benefitfrom treatment with an anti-CD70 binding agent, as described herein. A“CD70-associated disorder” and “CD70-associated disease” typicallyexpress CD70, or a fragment thereof, on the cell surface. This includeschronic and acute disorders or diseases including those pathologicalconditions that predispose the mammal to the disorder in question.Non-limiting examples or disorders to be treated herein include cancer,hematological malignancies, benign and malignant tumors, leukemias andlymphoid malignancies, carcinomas, and inflammatory, angiogenic andimmunologic disorders. Specific examples of disorders are disclosedinfra.

The terms “treatment” and “therapy”, and the like, as used herein, aremeant to include therapeutic as well as prophylactic, or suppressivemeasures for a disease or disorder leading to any clinically desirableor beneficial effect, including but not limited to alleviation or reliefof one or more symptoms, regression, slowing or cessation of progressionof the disease or disorder. Thus, for example, the term treatmentincludes the administration of an agent prior to or following the onsetof a symptom of a disease or disorder, thereby preventing or removingall signs of the disease or disorder. As another example, the termincludes the administration of an agent after clinical manifestation ofthe disease to combat the symptoms of the disease. Further,administration of an agent after onset and after clinical symptoms havedeveloped where administration affects clinical parameters of thedisease or disorder, such as the degree of tissue injury or the amountor extent of metastasis, whether or not the treatment leads toamelioration of the disease, comprises “treatment” or “therapy” as usedherein.

As used herein, the terms “prevention” or “prevent” refer toadministration of an anti-CD70 binding agent to a subject before theonset of a clinical or diagnostic symptom of a CD70-expressing cancer orimmunological disorder (e.g., administration to an individual with apredisposition or at a high risk of acquiring the CD70-expressing canceror immunological disorder) to (a) block the occurrence or onset of theCD70-expressing cancer or immunological disorder, or one or more ofclinical or diagnostic symptoms thereof, (b) inhibit the severity ofonset of the CD70-expressing cancer or immunological disorder, or (c) tolessen the likelihood of the onset of the CD70-expressing cancer orimmunological disorder.

The term “intravenous infusion” refers to introduction of an agent,e.g., a therapeutic agent, into the vein of an animal or human patientover a period of time greater than approximately 15 minutes, generallybetween approximately 30 to 90 minutes.

The term “intravenous bolus” or “intravenous push” refers to drugadministration into a vein of an animal or human such that the bodyreceives the drug in approximately 15 minutes or less, generally 5minutes or less.

The term “subcutaneous administration” refers to introduction of anagent, e.g., a therapeutic agent, under the skin of an animal or humanpatient, typically within a pocket between the skin and underlyingtissue, by relatively slow, sustained delivery from a drug receptacle.Pinching or drawing the skin up and away from underlying tissue maycreate the pocket.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as an antibody) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human patient, where bolus drug delivery is lessthan approximately 15 minutes; in another aspect, less than 5 minutes,and in still another aspect, less than 60 seconds. In yet even anotheraspect, administration is within a pocket between the skin andunderlying tissue, where the pocket may be created by pinching ordrawing the skin up and away from underlying tissue.

The term “effective amount” refers to the amount of an anti-CD70 bindingagent (e.g., an antibody or derivative or other binding agent) that issufficient to inhibit the occurrence or ameliorate one or more clinicalor diagnostic symptoms of a CD70-expressing cancer or immunologicaldisorder in a subject. An effective amount of an agent is administeredaccording to the methods described herein in an “effective regimen.” Theterm “effective regimen” refers to a combination of amount of the agentand dosage frequency adequate to accomplish treatment or prevention of aCD70-expressing cancer or immunological disorder.

The term “therapeutically effective amount” is used to refer to anamount of a therapeutic agent having beneficial patient outcome, forexample, a growth arrest effect or deletion of the cell. In one aspect,the therapeutically effective amount has apoptotic activity, or iscapable of inducing cell death. In another aspect, the therapeuticallyeffective amount refers to a target serum concentration that has beenshown to be effective in, for example, slowing disease progression.Efficacy can be measured in conventional ways, depending on thecondition to be treated. For example, in neoplastic diseases ordisorders characterized by cells expressing CD70, efficacy can bemeasured by assessing the time to disease progression (TTP), ordetermining the response rates (RR).

The term “pharmaceutically acceptable” as used herein means approved bya regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. The term “pharmaceuticallycompatible ingredient” refers to a pharmaceutically acceptable diluent,adjuvant, excipient, or vehicle with which an anti-CD70-binding agent isadministered.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of an anti-CD70binding agent or therapeutic agent. The anti-CD70 binding agent ortherapeutic agent contains at least one amino group, and accordinglyacid addition salts can be formed with this amino group or othersuitable groups. Exemplary salts include, but are not limited, tosulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p toluenesulfonate,and pamoate (i.e., 1,1′ methylene bis-(2 hydroxy 3 naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

“Pharmaceutically acceptable solvate” or “solvate” refer to anassociation of one or more solvent molecules and an anti-CD70 bindingagent and/or therapeutic agent. Examples of solvents that formpharmaceutically acceptable solvates include, but are not limited to,water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,and ethanolamine.

The abbreviation “AFP” refers todimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine.

The abbreviation “MMAE” refers to monomethyl auristatin E.

The abbreviation “AEB” refers to an ester produced by reactingauristatin E with paraacetyl benzoic acid.

The abbreviation “AEVB” refers to an ester produced by reactingauristatin E with benzoylvaleric acid.

The abbreviation “MMAF” refers todovaline-valine-dolaisoleunine-dolaproine-phenylalanine.

The abbreviations “fk” and “phe-lys” refer to the linkerphenylalanine-lysine.

II. ANTI-CD70 ANTIBODIES AND DERIVATIVES THEREOF

The compositions and methods described herein encompass the use of aCD70 binding agent that specifically binds to CD70. The CD70 bindingagent may exert a cytotoxic, cytostatic or immunomodulatory effect onCD70-expressing cancer cells, activated immune cells or other targetcells. The CD70 binding agent can be, for example, an anti-CD70antibody, an antigen-binding fragment of an anti-CD70 antibody, aderivative thereof, or other CD70-binding agent comprising at least onecomplementarity determining region (CDR) of a CD70-binding antibody.

In one aspect, the CD70 binding agent comprises one or morecomplementarity determining regions (CDRs) identical, substantiallyidentical or substantially similar to one or more CDR(s) of monoclonalantibody 1F6. (The nucleic acid and amino acid sequences of the heavyand light chain variable regions of 1F6 are set forth in SEQ ID NO:1 andSEQ ID NO:2, and SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and aredisclosed in International Patent Publication No. WO 04/073656; thedisclosure of which is incorporated by reference herein.) For example,the binding agent can include a heavy chain CDR and/or a light chain CDRthat is identical or substantially identical or substantially similar toa corresponding heavy chain CDR (H1, H2, or H3 regions) or correspondinglight chain CDR (L1, L2, or L3 regions) of mAb 1F6. In typicalembodiments, the anti-CD70 binding agent has two or three heavy chainCDRs and/or two or three light chain CDRs that are identical,substantially identical or substantially similar to corresponding heavyand/or light chain CDRs of mAb 1F6.

For example, in some embodiments, where the anti-CD70 binding agent hasat least one heavy chain CDR substantially identical or substantiallysimilar to a heavy chain CDR of mAb 1F6, the binding agent can furtherinclude at least one light chain CDR that is substantially identical orsubstantially similar to a light chain CDR of mAb 1F6.

In some embodiments, the anti-CD70 binding agent includes a heavy orlight chain variable domain, the variable domain having (a) a set ofthree CDRs identical, substantially identical or substantially similarto corresponding CDRs of mAb 1F6, and (b) a set of four variable regionframework regions from a human immunoglobulin. For example, an anti-CD70antibody can include a heavy and/or light chain variable domain(s), thevariable domain(s) having (a) a set of three CDRs, in which the set ofCDRs are from monoclonal antibody 1F6, and (b) a set of four frameworkregions derived from a human IgG. The antibody can optionally include ahinge region. In an exemplary embodiment, the anti-CD70 antibody is afully humanized antibody.

In another aspect, the CD70 binding agent comprises one or morecomplementarity determining regions (CDRs) substantially identical orsubstantially similar to one or more CDR(s) of monoclonal antibody 2F2.(The nucleic acid and amino acid sequences of the heavy and light chainvariable regions of 2F2 are set forth in SEQ ID NO:27 and SEQ ID NO:28,and SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and are disclosed inInternational Patent Publication No. WO 04/073656; the disclosure ofwhich is incorporated by reference herein.) For example, the bindingagent can include a heavy chain CDR and/or a light chain CDR that isidentical or substantially identical or substantially similar to acorresponding heavy chain CDR (H1, H2, or H3 regions) or correspondinglight chain CDR (L1, L2, or L3 regions) of mAb 2F2. In typicalembodiments, the anti-CD70 binding agent has two or three heavy chainCDRs and/or two or three light chain CDRs that are identical,substantially identical or substantially similar to corresponding heavyand/or light chain CDRs of mAb 2F2.

For example, in some embodiments, where an anti-CD70 antibody has atleast one heavy chain CDR substantially identical or substantiallysimilar to a heavy chain CDR of mAb 2F2, the antibody or derivativethereof can further include at least one light chain CDR that issubstantially identical or substantially similar to a light chain CDR ofmAb 2F2.

In some embodiments, the anti-CD70 binding agent includes a heavy orlight chain variable domain, the variable domain having (a) a set ofthree CDRs identical, substantially identical or substantially similarto corresponding CDRs of mAb 2F2, and (b) a set of four variable regionframework regions from a human immunoglobulin. For example, an anti-CD70antibody can include a heavy and/or light chain variable domain(s), thevariable domain(s) having (a) a set of three CDRs, in which the set ofCDRs are from monoclonal antibody 2F2, and (b) a set of four frameworkregions derived from a human IgG. The antibody can optionally include ahinge region. In an exemplary embodiment, the anti-CD70 antibody is afully humanized antibody.

In some embodiments, the framework regions are chosen from humangermline exon V_(H), J_(H), Vκ and Jκ sequences. For example, acceptorsequences for humanization of FR of a c1F6 V_(H) domain can be chosenfrom germline V_(H) exons V_(H)1-18 (Matsuda et al., 1993, NatureGenetics 3:88-94) or V_(H)1-2 (Shin et al., 1991, EMBO J. 10:3641-3645)and for the hinge region (J_(H)), exon J_(H)-6 (Mattila et al., 1995,Eur. J. Immunol. 25:2578-2582). In other examples, germline Vκ exon B3(Cox et al., 1994, Eur. J. Immunol. 24:827-836) and Jκ exon Jκ-1 (Hieteret al., 1982, J. Biol. Chem. 257:1516-1522) can be chosen as acceptorsequences for c1F6 V_(L) domain humanization.

In some embodiments, the sequence of the framework region of thehumanized anti-CD70 antibody includes a derivative of the acceptor humangermline exon used, including derivatives in which mouse donor residuesare reintroduced. These residues include reintroduction of the mousedonor residue at one or more of positions H46, H67, H68, H69, H70, H71,H80, H81, H82, H82A and H91 in the V_(H) domain, according to the Kabatnumbering convention.

The following table indicates the regions of humanized 1F6 to which eachSEQ ID NO. corresponds.

TABLE 1 NUCLEOTIDE SEQ MOLECULE OR AMINO ACID ID NO c1F6 Heavy ChainVariable Region Nucleotide 1 c1F6 Heavy Chain Variable Region Amino Acid2 h1F6 hV_(H)-D + hIgG₁ Constant Domain Nucleotide 3 h1F6 hV_(H)-D +hIgG₁ Constant Domain Amino Acid 4 h1F6 hV_(H)-E Nucleotide 5 h1F6hV_(H)-E Amino Acid 6 h1F6 hV_(H)-E + hIgG₁ Constant Domain Nucleotide 7h1F6 hV_(H)-E + hIgG₁ Constant Domain Amino Acid 8 h1F6 hV_(H)-HNucleotide 9 h1F6 hV_(H)-H Amino Acid 10 h1F6 hV_(H)-H + hIgG₁ ConstantDomain Nucleotide 11 h1F6 hV_(H)-H + hIgG₁ Constant Domain Amino Acid 12h1F6 hV_(H)-J Nucleotide 13 h1F6 hV_(H)-J Amino Acid 14 h1F6 hV_(H)-J +hIgG₁ Constant Domain Nucleotide 15 h1F6 hV_(H)-J + hIgG₁ ConstantDomain Amino Acid 16 h1F6 hV_(H)-M Nucleotide 17 h1F6 hV_(H)-M AminoAcid 18 h1F6 hV_(H)-M + hIgG₁ Constant Domain Nucleotide 19 h1F6hV_(H)-M + hIgG₁ Constant Domain Amino Acid 20 c1F6 Light Chain VariableRegion Nucleotide 21 c1F6 Light Chain Variable Region Amino Acid 22hV_(L)A Nucleotide 23 hV_(L)A Amino Acid 24 hV_(L)A + human κ constantdomain Nucleotide 25 hV_(L)A + human κ constant domain Amino Acid 26c2F2 Heavy Chain Variable Region Nucleotide 27 c2F2 Heavy Chain VariableRegion Amino Acid 28 c2F2 Light Chain Variable Region Nucleotide 29 c2F2Light Chain Variable Region Amino Acid 30

In some embodiments, the CD70 binding agent can be a humanized antibodyor antigen-binding fragment of antibody 1F6 or 2F2. In some embodiments,the antibody or antigen-binding fragment comprises a polypeptide chainhaving the amino acid sequence of SEQ ID NO:6, SEQ ID NO:10, SEQ IDNO:14, SEQ ID NO:18, or amino acids 20-137 of SEQ ID NO:4. In someembodiments, the antibody or antigen-binding fragment comprises apolypeptide chain having the amino acid sequence of SEQ ID NO:24.

In some embodiments, the antibody or antigen-binding fragment comprisesa polypeptide chain that is at least 80% identical to the amino acidsequence of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, oramino acids 20-137 of SEQ ID NO:4. In some embodiments, the antibody orantigen-binding fragment comprises a polypeptide chain that is at least85% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:10,SEQ ID NO:14, SEQ ID NO:18, or amino acids 20-137 of SEQ ID NO:4. Insome embodiments, the antibody or antigen-binding fragment comprises apolypeptide chain that is at least 90% identical to the amino acidsequence of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, oramino acids 20-137 of SEQ ID NO:4. In some embodiments, the antibody orantigen-binding fragment comprises a polypeptide chain that is at least95% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:10,SEQ ID NO:14, SEQ ID NO:18, or amino acids 20-137 of SEQ ID NO:4. Insome embodiments, the antibody or antigen-binding fragment comprises apolypeptide chain that is at least 99% identical to the amino acidsequence of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, oramino acids 20-137 of SEQ ID NO:4. In some embodiments, the polypeptidedoes not have the amino acid sequence of the heavy chain variable regionof antibody 1F6 or 2F2.

In some embodiments, the antibody or antigen-binding fragment comprisesa polypeptide chain that is at least 80% identical to the amino acidsequence of SEQ ID NO:24. In some embodiments, the antibody orantigen-binding fragment comprises a polypeptide chain that is at least85% identical to the amino acid sequence of SEQ ID NO:24. In someembodiments, the antibody or antigen-binding fragment comprises apolypeptide chain that is at least 90% identical to the amino acidsequence of SEQ ID NO:24. In some embodiments, the antibody orantigen-binding fragment comprises a polypeptide chain that is at least95% identical to the amino acid sequence of SEQ ID NO:24. In someembodiments, the antibody or antigen-binding fragment comprises apolypeptide chain that is at least 99% identical to the amino acidsequence of SEQ ID NO:24. In some embodiments, the polypeptide does nothave the amino acid sequence of the light chain variable region ofantibody 1F6 or 2F2.

In some embodiments, the anti-CD70 binding agent competes withmonoclonal antibody 1F6 or 2F2 for binding to human CD70. In someembodiments, the CD70 binding agent does not induce an agonistic orantagonistic signal when binding to CD70 (e.g., does not stimulateproliferation). In some embodiments, the CD70 binding agent blocksbinding of CD27 to CD70 by at least 20%, at least 30%, at least 40%, atleast 50%, at least 60, at least 70%, at least 80% or at least 90%.

The CD70-binding agent can optionally include an antibody effectordomain that mediates or stimulates an ADCC, ADCP and/or CDC responseagainst a CD70-expressing target cell. The effector domain(s) can be,for example, an Fc domain or domains of an Ig molecule. Such aCD70-binding agent can exert a cytotoxic or cytostatic effect onCD70-expressing cancer cells, or exert a cytotoxic, cytostatic, orimmunomodulatory effect on activated lymphocytes or dendritic cells, forexample, in the treatment of a CD70-expressing cancer or animmunological disorder, respectively. Typically, the CD70-binding agentrecruits and/or activates cytotoxic white blood cells (e.g., naturalkiller (NK) cells, phagocytotic cells (e.g., macrophages), and/or serumcomplement components).

The anti-CD70 antibody can be a humanized antibody, a single chainantibody, an scFv, a diabody, an Fab, a minibody, an scFv-Fc, an Fv, orthe like. In some embodiments, a CD70 antigen-binding region can bejoined to an effector domain or domains such as, for example, thehinge-C_(H)2-C_(H)3 domains of an immunoglobulin, or a portion orfragment of an effector domain(s) having effector function.Antigen-binding antibody fragments, including single-chain antibodies,can comprise, for example, the variable region(s) in combination withthe entirety or a portion of an effector domain (e.g., a C_(H)2 and/orC_(H)3 domain alone or in combination with a C_(H)1, hinge and/or C_(L)domain). Also, antigen-binding fragments can comprise any combination ofeffector domains. In some embodiments, the anti-CD70 antibody can be asingle chain antibody comprising a CD70-binding variable region joinedto hinge-C_(H)2-C_(H)3 domains.

The effector domains of the anti-CD70 antibody can be from any suitablehuman immunoglobulin isotype. For example, the ability of humanimmunoglobulin to mediate CDC and ADCC/ADCP is generally in the order ofIgM≈IgG1≈IgG3>IgG2>IgG4 and IgG1≈IgG3>IgG2/IgM/IgG4, respectively. ACD70-binding polypeptide can be expressed as a recombinant fusionprotein comprising of the appropriate constant domains to yield thedesired effector function(s). Upon binding to target cells, theanti-CD70 antibodies or derivatives can trigger in vitro and in vivotarget cell destruction through an antibody effector function, such asADCC, CDC, and ADCP.

The CD70-binding agent optionally can be conjugated to a therapeuticagent, such as a cytotoxic, cytostatic or immunomodulatory agent.Suitable therapeutic agents are described herein.

In some embodiments, an anti-CD70 antibody can be chimeric, comprising ahuman or non-human Fc region or portion thereof. For example, theantibody can include a Fc domain or portion of non-human origin, e.g.,rodent (e.g., mouse or rat), donkey, sheep, rabbit, goat, guinea pig,camelid, horse, chicken or monkey (e.g., macaque, rhesus or the like).

An anti-CD70 binding agent, such as an antibody, can be monospecific,bispecific, trispecific, or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of CD70 and/or may bespecific for both CD70 as well as for a heterologous protein. (See,e.g., PCT Publications WO 93/17715, WO 92/08802, WO 91/00360, and WO92/05793; Tutt et al., 1991, J. Immunol. 147:60-69; U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; Kostelny etal., 1992, J. Immunol. 148:1547-1553.) Multispecific antibodies,including bispecific and trispecific antibodies, useful for practicingthe methods described herein are antibodies that immunospecifically bindto both CD70 (including but not limited to antibodies that have the CDRsof the monoclonal antibodies 2F2 and 1F6) and a second cell surfacereceptor or receptor complex that mediates ADCC, ADCP, and/or CDC, suchas CD16/FcγRIII, CD64/FcγRI, killer inhibitory or activating receptors,or the complement control protein CD59. In some embodiments, the bindingof the portion of the multispecific antibody to the second cell surfacemolecule or receptor complex may enhance the effector functions of theanti-CD70 antibody or other CD70 binding agent.

Anti-CD70 antibodies and derivatives thereof and other binding agentsmay also be described or specified in terms of their binding affinity toCD70. Typical binding affinities include those with a dissociationconstant or Kd less than 5×10⁻²M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M,10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M,10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, 10⁻¹² M, 5×⁻¹³ M, 5×10⁻¹⁴ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M,or 10⁻¹⁵ M.

The antibodies can be generated by methods known in the art. Forexample, monoclonal antibodies can be prepared using a wide variety oftechniques including, e.g., the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. Hybridoma techniques aregenerally discussed in, for example, Harlow et al., Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988);and Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas,pp. 563-681 (Elsevier, N.Y., 1981). Examples of phage display methodsthat can be used to make the anti-CD70 antibodies include, e.g., thosedisclosed in Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Markset al., 1991, J. Mol. Biol. 222:581; Quan and Carter, 2002, The rise ofmonoclonal antibodies as therapeutics in Anti-IgE and Allergic Disease,Jardieu and Fick Jr., eds., Marcel Dekker, New York, N.Y., Chapter 20,pp. 427-469; Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ameset al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al.,1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18;Burton et al., 1994, Advances in Immunology 57:191-280; PCT ApplicationNo. PCT/GB91/01134; PCT Publications WO 90/02809, WO 91/10737, WO92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and U.S.Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908;5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;5,658,727; 5,733,743 and 5,969,108 (the disclosures of which areincorporated by reference herein).

Examples of techniques that can be used to produce single-chainantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., 1991, Methods in Enzymology 203:46-88; Shu etal., 1993, Proc. Natl. Acad. Sci. USA 90:7995-7999; and Skerra et al.,1988, Science 240:1038-1040.

Methods for making bispecific antibodies are known in the art.Traditional production of full-length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (see, e.g., Milsteinet al., 1983, Nature 305:537-39). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichsome have the correct bispecific structure. Similar procedures aredisclosed in International Publication No. WO 93/08829, and inTraunecker et al., 1991, EMBO J. 10:3655-59.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion typicallyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H)2, and C_(H)3 regions. In someembodiments, the fusion includes a first heavy-chain constant region(C_(H)1) containing the site necessary for light chain binding, presentin at least one of the fusions. Nucleic acids with sequences encodingthe immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In an embodiment of this approach, the bispecific antibodies have ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. Thisasymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation (see, e.g.,International Publication No. WO 94/04690, which is incorporated hereinby reference in its entirety).

For further discussion of bispecific antibodies see, for example, Sureshet al., 1986, Methods in Enzymology 121:210; Rodrigues et al., 1993, J.Immunology 151:6954-61; Carter et al., 1992, Bio/Technology 10:163-67;Carter et al., 1995, J. Hematotherapy 4:463-70; Merchant et al., 1998,Nature Biotechnology 16:677-81. Using such techniques, bispecificantibodies can be prepared for use in the treatment or prevention ofdisease as defined herein.

Bifunctional antibodies are also described in European PatentPublication No. EPA 0 105 360. As disclosed in this reference, hybrid orbifunctional antibodies can be derived either biologically, i.e., bycell fusion techniques, or chemically, especially with cross-linkingagents or disulfide-bridge forming reagents, and may comprise wholeantibodies or fragments thereof. Methods for obtaining such hybridantibodies are disclosed for example in International Publication WO83/03679 and European Patent Publication No. EPA 0 217 577, both ofwhich are incorporated herein by reference.

In some embodiments, framework residues in the human framework regionswill be substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., U.S. Pat. No. 5,585,089; Riechmann et al., 1988,Nature 332:323.) Antibodies can be humanized using a variety oftechniques known in the art including, for example, CDR-grafting (see,e.g., EP 0 239 400; PCT Publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (see,e.g., EP 0 592 106; EP 0 519 596; Padlan, 1991, Molecular Immunology28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973), and chain shuffling (see, e.g., U.S. Pat. No. 5,565,332)(all of these references are incorporated by reference herein).

Humanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inInternational Publication No. WO 87/02671; European Patent PublicationNo. 0 184 187; European Patent Publication No. 0 171 496; EuropeanPatent Publication No. 0 173 494; International Publication No. WO86/01533; U.S. Pat. No. 4,816,567; European Patent Publication No. 0 012023; Berter et al., 1988, Science 240:1041-43; Liu et al., 1987, Proc.Natl. Acad. Sci. USA 84:3439-43; Liu et al., 1987, J. Immunol.139:3521-26; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-18;Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985,Nature 314:446-449; Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-59;Morrison, 1985, Science 229:1202-07; Oi et al., 1986, BioTechniques4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-25;Verhoeyan et al., 1988, Science 239:1534; and Beidler et al., 1988, J.Immunol. 141:4053-60; each of which is incorporated herein by referencein its entirety.

As set forth supra, a CD70 binding agent can be a derivative of ananti-CD70 antibody. Generally, an anti-CD70 antibody derivativecomprises an anti-CD70 antibody (including e.g., an antigen-bindingfragment or conservatively substituted polypeptides) and at least onepolypeptide region or other moiety heterologous to the anti-CD70antibody. For example, an anti-CD70 antibody can be modified, e.g., bythe covalent attachment of any type of molecule. Typical modificationsinclude, e.g., glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand (e.g., analbumin-binding molecule) or other protein, and the like. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc.

In some embodiments, the covalent attachment does not interfere witheffector function, e.g., prevent the antibody derivative fromspecifically binding to CD70 via the antigen-binding region or regionderived therefrom, or the effector domains(s) from specifically bindingFc receptor.

In some embodiments, the antibody derivative is a multimer, such as, forexample, a dimer, comprising one or more monomers, where each monomerincludes (i) an antigen-binding region of an anti-CD70 antibody, or apolypeptide region derived therefrom (such as, e.g., by conservativesubstitution of one or more amino acids), and (ii) a multimerizing(e.g., dimerizing) polypeptide region, such that the antibody derivativeforms multimers (e.g., homodimers) that specifically bind to CD70. Intypical embodiments, an antigen-binding region of an anti-CD70 antibody,or a polypeptide region derived therefrom, is recombinantly orchemically fused with a heterologous protein, wherein the heterologousprotein comprises a dimerization or multimerization domain. Prior toadministration of the antibody derivative to a subject for the purposeof treating or preventing immunological disorders or CD70-expressingcancers, the derivative is subjected to conditions that allow formationof a homodimer or heterodimer. A heterodimer, as used herein, maycomprise identical dimerization domains but different CD70antigen-binding regions, identical CD70 antigen-binding regions butdifferent dimerization domains, or different CD70 antigen-bindingregions and dimerization domains.

Typical dimerization domains are those that originate from transcriptionfactors. In one embodiment, the dimerization domain is that of a basicregion leucine zipper (“bZIP”) (see Vinson et al., 1989, Science246:911-916). Useful leucine zipper domains include, for example, thoseof the yeast transcription factor GCN4, the mammalian transcriptionfactor CCAAT/enhancer-binding protein C/EBP, and the nuclear transformin oncogene products, Fos and Jun. (See, e.g., Landschultz et al., 1988,Science 240:1759-64; Baxevanis and Vinson, 1993, Curr. Op. Gen. Devel.3:278-285; O'Shea et al., 1989, Science 243:538-542.) In anotherembodiment, the dimerization domain is that of a basic-regionhelix-loop-helix (“bHLH”) protein. (See, e.g., Murre et al., 1989, Cell56:777-783. See also Davis et al., 1990, Cell 60:733-746; Voronova andBaltimore, 1990, Proc. Natl. Acad. Sci. USA 87:4722-26.) Particularlyuseful hHLH proteins are myc, max, and mac.

In yet other embodiments, the dimerization domain is an immunoglobulinconstant region such as, for example, a heavy chain constant region or adomain thereof (e.g., a C_(H)1 domain, a C_(H)2 domain, and/or a C_(H)3domain). (See, e.g., U.S. Pat. Nos. 5,155,027; 5,336,603; 5,359,046; and5,349,053; EP 0 367 166; and WO 96/04388.)

Heterodimers are known to form between Fos and Jun (Bohmann et al.,1987, Science 238:1386-1392), among members of the ATF/CREB family (Haiet al., 1989, Genes Dev. 3:2083-2090), among members of the C/EBP family(Cao et al., 1991, Genes Dev. 5:1538-52; Williams et al., 1991, GenesDev. 5:1553-67; Roman et al., 1990, Genes Dev. 4:1404-15), and betweenmembers of the ATF/CREB and Fos/Jun families (Hai and Curran, 1991,Proc. Natl. Acad. Sci. USA 88:3720-24). Therefore, when a CD70-bindingprotein is administered to a subject as a heterodimer comprisingdifferent dimerization domains, any combination of the foregoing may beused.

In other embodiments, an anti-CD70 antibody derivative is an anti-CD70antibody conjugated to a second antibody (an “antibody heteroconjugate”)(see, e.g., U.S. Pat. No. 4,676,980). Heteroconjugates useful forpracticing the present methods comprise an antibody that binds to CD70(e.g., an antibody that has the CDRs and/or heavy chains of themonoclonal antibodies 2F2 or 1F6) and an antibody that binds to asurface receptor or receptor complex that mediates ADCC, phagocytosis,and/or CDC, such as CD16/FcgRIII, CD64/FcgRI, killer cell activating orinhibitory receptors, or the complement control protein CD59. In atypical embodiment, the binding of the portion of the multispecificantibody to the second cell surface molecule or receptor complexenhances the effector functions of an anti-CD70 antibody. In otherembodiments, the antibody can be a therapeutic agent. Suitable antibodytherapeutic agents are described herein.

In some embodiments, the anti-CD70 antibody or derivative thereofcompetitively inhibits binding of mAb 1F6 or 2F2 to CD70, as determinedby any method known in the art for determining competitive binding (suchas e.g., the immunoassays described herein). In typical embodiments, theantibody competitively inhibits binding of 1F6 or 2F2 to CD70 by atleast 50%, at least 60%, at least 70%, or at least 75%. In otherembodiments, the antibody competitively inhibits binding of 1F6 or 2F2to CD70 by at least 80%, at least 85%, at least 90%, or at least 95%.

Antibodies can be assayed for specific binding to CD70 by any of variousknown methods. Immunoassays which can be used include, for example,competitive and non-competitive assay systems using techniques such asWestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, and protein A immunoassays. Suchassays are routine and well-known in the art. (See, e.g., Ausubel etal., eds., Short Protocols in Molecular Biology (John Wiley and Sons,Inc., New York, 4th ed. 1999); Harlow and Lane, Using Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1999.)

Further, the binding affinity of an antibody to CD70 and the off-rate ofan antibody CD70 interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled CD70 (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledCD70, and the detection of the antibody bound to the labeled CD70. Theaffinity of the antibody for CD70 and the binding off-rates can then bedetermined from the data by Scatchard plot analysis. Competition with asecond antibody (such as e.g., mAb 1F6 or 2F2) can also be determinedusing radioimmunoassays. In this case, CD70 is incubated with theantibody of interest conjugated to a labeled compound (e.g., ³H or ¹²⁵I)in the presence of increasing amounts of an unlabeled second antibody.Alternatively, the binding affinity of an antibody to CD70 and the on-and off-rates of an antibody-CD70 interaction can be determined bysurface plasmon resonance. In some embodiments, the anti-CD70 antibodiesor derivatives thereof can be targeted to and accumulate on the membraneof a CD70-expressing cell.

Anti-CD70 antibodies and derivatives thereof can be produced by methodsknown in the art for the synthesis of proteins, typically, e.g., byrecombinant expression techniques. Recombinant expression of an antibodyor derivative thereof that binds to CD70 typically includes constructionof an expression vector containing a nucleic acid that encodes theantibody or derivative thereof. A vector for the production of theprotein molecule may be produced by recombinant DNA technology usingtechniques known in the art. Standard techniques such as, for example,those described in Sambrook and Russell, Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,3rd ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2nd ed.,1989); Short Protocols in Molecular Biology (Ausubel et al., John Wileyand Sons, New York, 4th ed., 1999); and Glick and Pasternak, MolecularBiotechnology: Principles and Applications of Recombinant DNA (ASMPress, Washington, D.C., 2nd ed., 1998) can be used for recombinantnucleic acid methods, nucleic acid synthesis, cell culture, transgeneincorporation, and recombinant protein expression.

For example, for recombinant expression of an anti-CD70 antibody, anexpression vector may encode a heavy or light chain thereof, or a heavyor light chain variable domain, operably linked to a promoter. Anexpression vector may include, for example, the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464), and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain. Theexpression vector is transferred to a host cell by conventionaltechniques, and the transfected cells are then cultured by conventionaltechniques to produce the anti-CD70 antibody. In typical embodiments forthe expression of double-chained antibodies, vectors encoding both theheavy and light chains can be co-expressed in the host cell forexpression of the entire immunoglobulin molecule.

A variety of prokaryotic and eukaryotic host-expression vector systemscan be utilized to express an anti-CD70 antibody or derivative thereof.Typically, eukaryotic cells, particularly for whole recombinantanti-CD70 antibody molecules, are used for the expression of therecombinant protein. For example, mammalian cells such as Chinesehamster ovary cells (CHO), in conjunction with a vector such as themajor intermediate early gene promoter element from humancytomegalovirus, is an effective expression system for the production ofanti-CD70 antibodies and derivatives thereof (see, e.g., Foecking etal., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).

Other host-expression systems include, for example, plasmid-basedexpression systems in bacterial cells (see, e.g., Ruther et al., 1983,EMBO 1, 2:1791; Inouye and Inouye, 1985, Nucleic Acids Res.13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem.24:5503-5509); insect systems such as, e.g., the use of Autographacalifornica nuclear polyhedrosis virus (AcNPV) expression vector inSpodoptera frugiperda cells; and viral-based expression systems inmammalian cells, such as, e.g., adenoviral-based systems (see, e.g.,Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359; Bittner etal., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processing(e.g., glycosylation, phosphorylation, and cleavage) of the proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript and geneproduct can be used. Such mammalian host cells include, for example,CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138.

A stable expression system is typically used for long-term, high-yieldproduction of recombinant anti-CD70 antibody or derivative thereof orother CD70 binding agent. For example, cell lines that stably expressthe anti-CD70 antibody or derivative thereof can be engineered bytransformation of host cells with DNA controlled by appropriateexpression control elements (e.g., promoter and enhancer sequences,transcription terminators, polyadenylation sites) and a selectablemarker, followed by growth of the transformed cells in a selectivemedia. The selectable marker confers resistance to the selection andallows cells to stably integrate the DNA into their chromosomes and growto form foci which in turn can be cloned and expanded into cell lines. Anumber of selection systems can be used, including, for example, theherpes simplex virus thymidine kinase, hypoxanthineguaninephosphoribosyltransferase, and adenine phosphoribosyltransferase genes,which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate; gpt,which confers resistance to mycophenolic acid; neo, which confersresistance to the aminoglycoside G-418; and hygro, which confersresistance to hygromycin. Methods commonly known in the art ofrecombinant DNA technology can be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Current Protocols in Molecular Biology (Ausubel et al. eds., JohnWiley and Sons, N.Y., 1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual (Stockton Press, N.Y., 1990); Current Protocols inHuman Genetics (Dracopoli et al. eds., John Wiley and Sons, N.Y., 1994,Chapters 12 and 13); and Colberre-Garapin et al., 1981, J. Mol. Biol.150:1.

The expression levels of an antibody or derivative can be increased byvector amplification. (See generally, e.g., Bebbington and Hentschel,The Use of Vectors Based on Gene Amplification for the Expression ofCloned Genes in Mammalian Cells in DNA Cloning, Vol. 3 (Academic Press,New York, 1987).) When a marker in the vector system expressing ananti-CD70 antibody or derivative thereof is amplifiable, an increase inthe level of inhibitor present in host cell culture media will selecthost cells that have increased copy number of a marker gene conferringresistance to the inhibitor. The copy number of an associated antibodygene will also be increased, thereby increasing expression of theantibody or derivative thereof (see Crouse et al., 1983, Mol. Cell.Biol. 3:257).

Where the anti-CD70 antibody comprises both a heavy and a light chain orderivatives thereof, the host cell may be co-transfected with twoexpression vectors, the first vector encoding the heavy chain proteinand the second vector encoding the light chain protein. The two vectorsmay contain identical selectable markers which enable equal expressionof heavy and light chain proteins. Alternatively, a single vector may beused which encodes, and is capable of expressing, both heavy and lightchain proteins. In such situations, the light chain is typically placedbefore the heavy chain to avoid an excess of toxic free heavy chain (seeProudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an anti-CD70 antibody or derivative thereof has been produced(e.g., by an animal, chemical synthesis, or recombinant expression), itcan be purified by any suitable method for purification of proteins,including, for example, by chromatography (e.g., ion exchange oraffinity chromatography (such as, for example, Protein A chromatographyfor purification of antibodies having an intact Fc region)),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. An anti-CD70 antibody orderivative thereof can, for example, be fused to a marker sequence, suchas a peptide, to facilitate purification by affinity chromatography.Suitable marker amino acid sequences include, e.g., a hexa-histidinepeptide, such as the tag provided in a pQE vector (QIAGEN, Inc.,Chatsworth, Calif., 91311), and the “HA” tag, which corresponds to anepitope derived from the influenza hemagglutinin protein (Wilson et al.,1984, Cell 37:767), and the “flag” tag.

Once an anti-CD70 antibody or derivative thereof is produced, itsability to exert a cytostatic or cytotoxic effect on CD70-expressingcancer cells or an immunomodulatory effect on a CD70-expressing immunecell is determined by the methods described infra or as known in theart.

To minimize activity of the anti-CD70 antibody outside the activatedimmune cells or CD70-expressing cancer cells, an antibody thatspecifically binds to cell membrane-bound CD70, but not to soluble CD70,can be used, so that the anti-CD70 antibody is concentrated at the cellsurface of the activated immune cell or CD70-expressing cancer cell.

Typically, the anti-CD70 antibody or derivative is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). In some embodiments, the anti-CD70antibody or derivative is at least about 40% pure, at least about 50%pure, or at least about 60% pure. In some embodiments, the anti-CD70antibody or derivative is at least about 60-65%, 65-70%, 70-75%, 75-80%,80-85%, 85-90%, 90-95%, or 95-98% pure. In some embodiments, theanti-CD70 antibody or derivative is approximately 99% pure.

III. OTHER CD70-BINDING AGENTS

Further CD70-binding agents include fusion proteins (i.e., proteins thatare recombinantly fused or chemically conjugated, including bothcovalent and non-covalent conjugation) to heterologous proteins (oftypically at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100amino acids). Such CD70-binding agents can include a portion that bindsto CD70 and an immunoglobulin effector domain or a functional equivalentthereof. As used herein, a functional equivalent of immunoglobulineffector domain binds to an Fc receptor on an immune cell withphagocytic or lytic activity or by binding of an Fc effector domain(s)to components of the complement system. The fusion protein does notnecessarily need to be direct, but may occur through linker sequences.

For example, a CD70-binding agent can be produced recombinantly byfusing the coding region of one or more of the CDRs or the variableregion of an anti-CD70 antibody in frame with a sequence coding for aheterologous protein. The heterologous protein can include, for example,an effector domain, a functional equivalent thereof or other functionaldomain to provide one or more of the following characteristics: promotestable expression; provide a means of facilitating high yieldrecombinant expression; provide a cytostatic, cytotoxic orimmunomodulatory activity; and/or provide a multimerization domain.

In some embodiments, the CD70-binding agent can include one or more CDRsfrom an antibody that binds to CD70 and depletes or inhibits theproliferation of CD70-expressing cells alone, without conjugation to acytotoxic agent.

IV. METHODS TO IMPROVE EFFECTOR FUNCTIONS OF ANTI-CD70-TARGETING AGENTS

In some embodiments, the effector function of a CD70-binding agent canbe augmented by improving its effector functions using one or moreantibody engineering approaches known in the art. Illustrative,non-limiting examples for such approaches are provided below.

ADCC and ADCP are mediated through the interaction of cell-boundantibodies with Fcγ receptors (FcγR) expressed on effector cells. Boththe glycosylation status and primary amino acid sequence of the IgG Fcregion have functional effects on the Fcγ-FcγR interaction. A strongerFcγ-FcγR interaction is associated with better target cell killing byeffector cells.

Oligosaccharides covalently attached to the conserved Asn297 areinvolved in the Fc region of an IgG to bind FcγR (Lund et al., 1996, J.Immunol. 157:4963-69; Wright and Morrison, 1997, Trends Biotechnol.15:26-31). Engineering of this glycoform on IgG can significantlyimprove IgG-mediated ADCC. Addition of bisecting N-acetylglucosaminemodifications (Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davieset al., 2001, Biotech. Bioeng. 74:288-94) to this glycoform or removalof fucose (Shields et al., 2002, J. Biol. Chem. 277:26733-40; Shinkawaet al., 2003, J. Biol. Chem. 278:6591-604; Niwa et al., 2004, CancerRes. 64:2127-33) from this glycoform are two examples of IgG Fcengineering that improves the binding between IgG Fc and FcγR, therebyenhancing Ig-mediated ADCC activity.

A systemic substitution of solvent-exposed amino acids of human IgG1 Fcregion has generated IgG variants with altered FcγR binding affinities(Shields et al., 2001, J. Biol. Chem. 276:6591-604). When compared toparental IgG1, a subset of these variants involving substitutions atThr256/Ser298, Ser298/Glu333, Ser298/Lys334, or Ser298/Glu333/Lys334 toAla demonstrate increased in both binding affinity toward FcγR and ADCCactivity (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki etal., 2004, J. Mol. Biol. 336:1239-49).

Antibody-mediated CDC begins with the binding of C1q to cell bound IgGmolecules. Specific amino acid residues on human IgG1 responsible forC1q binding and species-specific differences of C1q binding have beenreported (Idusogie et al., 2000, J. Immunol. 164:4178-4184). Complementfixation activity of antibodies have been improved by substitutions atLys326 and Glu333; for example, such substitutions can improve both C1qbinding and CDC activity of the human IgG1 antibody rituximab (Idusogieet al., 2001, J. Immunol. 166:2571-2575). The same substitutions on ahuman IgG2 backbone can convert an antibody isotype that binds poorly toC1q and is severely deficient in complement activation activity to onethat can both bind C1q and mediate CDC (Idusogie et al., 2001, J.Immunol. 166:2571-75). Several other methods have also been applied toimprove complement fixation activity of antibodies. For example, thegrafting of an 18-amino acid carboxyl-terminal tail piece of IgM to thecarboxyl-termini of IgG greatly enhances their CDC activity. This isobserved even with IgG4, which normally has no detectable CDC activity(Smith et al., 1995, J. Immunol. 154:2226-36). Also, substituting Ser444located close to the carboxy-terminal of IgG1 heavy chain with Cysinduced tail-to-tail dimerization of IgG1 with a 200-fold increase ofCDC activity over monomeric IgG1 (Shopes et al., 1992, J. Immunol.148:2918-22). In addition, a bispecific diabody construct withspecificity for C1q also confers CDC activity (Kontermann et al., 1997,Nat. Biotech. 15:629-31).

The in vivo half-life of an antibody can also impact on its effectorfunctions. In some embodiments, it is desirable to increase or decreasethe half-life of an antibody to modify its therapeutic activities. FcRnis a receptor that is structurally similar to MHC Class I antigen thatnon-covalently associates with β2-microglobulin. FcRn regulates thecatabolism of IgGs and their transcytosis across tissues (Ghetie andWard, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002,Immunol. Res. 25:97-113). The IgG-FcRn interaction takes place at pH 6.0(pH of intracellular vesicles) but not at pH 7.4 (pH of blood); thisinteraction enables IgGs to be recycled back to the circulation (Ghetieand Ward, 2000, Ann. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002,Immunol. Res. 25:97-113). The region on human IgG₁ involved in FcRnbinding has been mapped (Shields et al., 2001, J. Biol. Chem.276:6591-604). Alanine substitutions at positions Pro238, Thr256,Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG₁ enhanceFcRn binding (Shields et al., 2001, J. Biol. Chem. 276:6591-604). IgG₁molecules harboring these substitutions are expected to have longerserum half-lives. Consequently, these modified IgG₁ molecules may beable to carry out their effector functions, and hence exert theirtherapeutic efficacies, over a longer period of time compared tounmodified IgG₁.

V. ASSAYS FOR CYTOTOXIC, CYTOSTATIC, AND IMMUNOMODULATORY ACTIVITIES

Methods of determining whether an antibody mediates effector functionagainst a target cell are known. Illustrative examples of such methodsare described infra.

For determining whether an anti-CD70 antibody or derivative mediatesantibody-dependent cellular cytotoxicity against activated immune cellsor CD70-expressing cancer cells, an assay that measures target celldeath in the presence of antibody and effector immune cells may be used.An assay used to measure this type of cytotoxicity can be based ondetermination of ⁵¹Cr release from metabolically-labeled targets cellsafter incubation in the presence of effector cells and target-specificantibody (see, e.g., Perussia and Loza, 2000, Methods in MolecularBiology 121:179-92; and “⁵¹Cr Release Assay of Antibody-DependentCell-Mediated Cytotoxicity (ADCC)” in Current Protocols in Immunology,Coligan et al. eds., Wileyand Sons, 1993). For example, activated immunecells (e.g., activated lymphocytes) or CD70-expressing cancer cellslabeled with Na₂ ⁵¹CrO₄ and plated at a density of 5,000 cells per wellof a 96-well plate can be treated with varying concentrations ofanti-CD70 antibody for 30 minutes then mixed with normal humanperipheral blood mononuclear cells (PBMC) for 4 hours. The membranedisruption that accompanies target cell death releases ⁵¹Cr into theculture supernatant which may be collected and assessed forradioactivity as a measure of cytotoxic activity. Other assays tomeasure ADCC may involve nonradioactive labels or be based on inducedrelease of specific enzymes. For example, a non-radioactive assay basedon time-resolved fluorometry is commercially available (Delphia, PerkinElmer). This assay is based on loading target cells with anacetoxymethyl ester of fluorescence enhancing ligand (BATDA) thatpenetrates the cell membrane then hydrolyses to form a membraneimpermeable hydrophilic ligand (TDA). When mixed with target specificantibody and PBMC effector cells, TDA is released from lysed cells andis available to form a highly fluorescent chelate when mixed withEuropium. The signal, measured with a time-resolved fluorometer,correlates with the amount of cell lysis.

To determine whether an anti-CD70 antibody or derivative mediatesantibody-dependent cellular phagocytosis against activated immune cellsor CD70-expressing cancer cells, an assay that measures target cellinternalization by effector immune cells (e.g., fresh culturedmacrophages or established macrophage-like cell line) may be used (see,e.g., Munn and Cheung, 1990, J. Exp. Med. 172:231-37; Keler et al.,2000, J. Immunol. 164:5746-52; Akewanlop et al., 2001, Cancer Res.61:4061-65). For example, target cells may be labeled with a lipophilicmembrane dye such as PKH67 (Sigma), coated with target-specificantibody, and mixed with effector immune cells for 4-24 hours. Theeffector cells may then be identified by counterstaining with afluorochrome-labeled antibody specific for a phagocytic cell surfacemarker (e.g., CD14) and the cells analyzed by two-color flow cytometryor fluorescence microscopy. Dual-positive cells represent effector cellsthat have internalized target cells. For these assays, effector cellsmay be monocytes derived from PBMC that have been differentiated intomacrophages by culture for 5-10 days with M-CSF or GM-CSF (see, e.g.,Munn and Cheung, supra). Human macrophage-like cell lines U937 (Larricket al., 1980, J. Immunology 125:6-12) or THP-1 (Tsuchiya et al., 1980,Int. J. Cancer 26:171-76) which are available from ATCC may be used asan alternative phagocytic cell source.

Methods of determining whether an antibody mediates complement-dependentcytotoxicity upon binding to target cells are also known. The samemethods can be applied to determine whether a CD70-binding agentmediates CDC on activated immune cells or CD70-expressing cancer cells.Illustrative examples of such methods are described infra.

The source of active complement can either be normal human serum orpurified from laboratory animal including rabbits. In a standard assay,a CD70-binding agent is incubated with CD70-expressing activated immunecells (e.g., activated lymphocytes) or CD70-expressing cancer cells inthe presence of complement. The ability of such CD70-binding agent tomediate cell lysis can be determined by several readouts. In oneexample, a Na⁵¹CrO₄ release assay is used. In this assay, target cellsare labeled with Na⁵¹CrO₄. Unincorporated Na⁵¹CrO₄ is washed off andcells are plated at a suitable density, typically between 5,000 to50,000 cells/well, in a 96-well plate. Incubation with the CD70-bindingagent in the presence of normal serum or purified complement typicallylast for 2-6 hours at 37° C. in a 5% CO₂ atmosphere. Releasedradioactivity, indicating cell lysis, is determined in an aliquot of theculture supernatant by gamma ray counting. Maximum cell lysis isdetermined by releasing incorporated Na⁵¹CrO₄ by detergent (0.5-1% NP-40or Triton X-100) treatment. Spontaneous background cell lysis isdetermined in wells where only complement is present without anyCD70-binding agents. Percentage cell lysis is calculated as(CD70-binding agent-induced lysis−spontaneous lysis)/maximum cell lysis.The second readout is a reduction of metabolic dyes, e.g., Alamar Blue,by viable cells. In this assay, target cells are incubated withCD70-binding agent with complement and incubated as described above. Atthe end of incubation, 1/10 volume of Alamar Blue (BiosourceInternational, Camarillo, Calif.) is added. Incubation is continued forup to 16 hours at 37° C. in a 5% CO₂ atmosphere. Reduction of AlamarBlue as an indication of metabolically active viable cells is determinedby fluorometric analysis with excitation at 530 nm and emission at 590nm. The third readout is cellular membrane permeability to propidiumiodide (PI). Formation of pores in the plasma membrane as a result ofcomplement activation facilitates entry of PI into cells where it willdiffuse into the nuclei and bind DNA. Upon binding to DNA, PIfluorescence in the 600 nm significantly increases. Treatment of targetcells with CD70-binding agent and complement is carried out as describedabove. At end of incubation, PI is added to a final concentration of 5μg/ml. The cell suspension is then examined by flow cytometry using a488 nm argon laser for excitation. Lysed cells are detected byfluorescence emission at 600 nm.

VI. ANIMAL MODELS OF IMMUNOLOGICAL DISORDERS OR CD70-EXPRESSING CANCERS

The anti-CD70 binding agents, e.g., antibodies or derivatives, can betested or validated in animal models of immunological disorders orCD70-expressing cancers. A number of established animal models ofimmunological disorders or CD70-expressing cancers are known to theskilled artisan, any of which can be used to assay the efficacy of theanti-CD70 antibody or derivative. Non-limiting examples of such modelsare described infra.

Examples for animal models of systemic and organ-specific autoimmunediseases including diabetes, lupus, systemic sclerosis, Sjögren'sSyndrome, experimental autoimmune encephalomyelitis (multiplesclerosis), thyroiditis, myasthenia gravis, arthritis, uveitis, andinflammatory bowel disease have been described by Bigazzi, “AnimalModels of Autoimmunity: Spontaneous and Induced,” in The AutoimmuneDiseases (Rose and Mackay eds., Academic Press, 1998) and in “AnimalModels for Autoimmune and Inflammatory Disease,” in Current Protocols inImmunology (Coligan et al. eds., Wiley and Sons, 1997).

Allergic conditions, e.g., asthma and dermatitis, can also be modeled inrodents. Airway hypersensitivity can be induced in mice by ovalbumin(Tomkinson et al., 2001, J. Immunol. 166:5792-800) or Schistosomamansoni egg antigen (Tesciuba et al., 2001, J. Immunol. 167:1996-2003).The Nc/Nga strain of mice show marked increase in serum IgE andspontaneously develop atopic dermatitis-like lesions (Vestergaard etal., 2000, Mol. Med. Today 6:209-10; Watanabe et al., 1997, Int.Immunol. 9:461-66; Saskawa et al., 2001, Int. Arch. Allergy Immunol.126:239-47).

Injection of immuno-competent donor lymphocytes into a lethallyirradiated histo-incompatible host is a classical approach to induceGVHD in mice. Alternatively, the parent B6D2F1 murine model provides asystem to induce both acute and chronic GVHD. In this model the B6D2F1mice are F1 progeny from a cross between the parental strains of C57BL/6and DBA/2 mice. Transfer of DBA/2 lymphoid cells into non-irradiatedB6D2F1 mice causes chronic GVHD, whereas transfer of C57BL/6, C57BL/10or B10.D2 lymphoid cells causes acute GVHD (Slayback et al., 2000, BoneMarrow Transpl. 26:931-938; Kataoka et al., 2001, Immunology103:310-318).

Additionally, both human hematopoietic stem cells and mature peripheralblood lymphoid cells can be engrafted into SCID mice, and these humanlympho-hematopoietic cells remain functional in the SCID mice (McCune etal., 1988, Science 241:1632-1639; Kamel-Reid and Dick, 1988, Science242:1706-1709; Mosier et al., 1988, Nature 335:256-259). This hasprovided a small animal model system for the direct testing of potentialtherapeutic agents on human lymphoid cells. (See, e.g., Tournoy et al.,2001, J. Immunol. 166:6982-6991).

Moreover, small animal models to examine the in vivo efficacies of theanti-CD70 antibodies or derivatives can be created by implantingCD70-expressing human tumor cell lines into appropriate immunodeficientrodent strains, e.g., athymic nude mice or SCID mice. Examples ofCD70-expressing human lymphoma cell lines include, for example, Daudi(Ghetie et al., 1994, Blood 83:1329-36; Ghetie et al., 1990, Int. J.Cancer 15:481-85; de Mont et al., 2001, Cancer Res. 61:7654-59),HS-Sultan (Cattan and Maung, 1996, Cancer Chemother. Pharmacol.38:548-52; Cattan and Douglas, 1994, Leuk. Res. 18:513-22), Raji(Ochakovskaya et al., 2001, Clin. Cancer Res. 7:1505-10; Breisto et al.,1999, Cancer Res. 59:2944-49), and CA46 (Kreitman et al., 1999, Int. J.Cancer 81:148-55). Non-limiting example of a CD70-expressing Hodgkin'slymphoma line is L428 (Drexler, 1993, Leuk. Lymphoma 9:1-25; Dewan etal., 2005, Cancer Sci. 96:466-473). Non-limiting examples of CD70expressing human renal cell carcinoma cell lines include 786-O (Ananthet al., 1999, Cancer Res. 59:2210-16; Datta et al., 2001, Cancer Res.61:1768-75), ACHN (Hara et al., 2001, J. Urol. 166:2491-94; Miyake etal., 2002, J. Urol. 167:2203-08), Caki-1 (Prewett et al., 1998, Clin.Cancer Res. 4:2957-66; Shi and Siemann, 2002, Br. J. Cancer 87:119-26),and Caki-2 (Zellweger et al., 2001, Neoplasia 3:360-67). Non-limitingexamples of CD70-expressing nasopharyngeal carcinoma cell lines includeC15 and C17 (Busson et al., 1988, Int. J. Cancer 42:599-606; Bernheim etal., 1993, Cancer Genet. Cytogenes. 66:11-5). Non-limiting examples ofCD70-expressing human glioma cell lines include U373 (Palma et al.,2000, Br. J. Cancer 82:480-7) and U87MG (Johns et al., 2002, Int. J.Cancer 98:398-408). Non-limiting examples of multiple myeloma cell linesinclude MM.1S (Greenstein et al., 2003, Experimental Hematology31:271-282) and L363 (Diehl et al., 1978, Blut 36:331-338). (See alsoDrexler and Matsuo, 2000, Leukemia Research 24:681-703). These tumorcell lines can be established in immunodeficient rodent hosts either assolid tumor by subcutaneous injections or as disseminated tumors byintravenous injections. Once established within a host, these tumormodels can be applied to evaluate the therapeutic efficacies of theanti-CD70 antibody or derivatives as described herein on modulating invivo tumor growth.

VII. CD70-ASSOCIATED DISORDERS

The anti-CD70 binding agents (e.g., antibodies and derivatives) asdescribed herein are useful for treating or preventing a CD70-expressingcancer or an immunological disorder characterized by expression of CD70by inappropriate activation of immune cells (e.g., lymphocytes ordendritic cells). Such expression of CD70 can be due to, for example,increased CD70 protein levels on the cells surface and/or alteredantigenicity of the expressed CD70. Treatment or prevention of theimmunological disorder, according to the methods described herein, isachieved by administering to a subject in need of such treatment orprevention an effective amount of the anti-CD70 antibody or derivative,whereby the antibody or derivative (i) binds to activated immune cellsthat express CD70 and that are associated with the disease state and(ii) exerts a cytotoxic, cytostatic, or immunomodulatory effect on theactivated immune cells. In some embodiments, the cytotoxic, cytostatic,or immunomodulatory is exerted without conjugation to a cytotoxic,cytostatic, or immunomodulatory agent. In some embodiments, thecytotoxic, cytostatic, or immunomodulatory is exerted by conjugation toa cytotoxic, cytostatic, or immunomodulatory agent.

Immunological diseases that are characterized by inappropriateactivation of immune cells and that can be treated or prevented by themethods described herein can be classified, for example, by the type(s)of hypersensitivity reaction(s) that underlie the disorder. Thesereactions are typically classified into four types: anaphylacticreactions, cytotoxic (cytolytic) reactions, immune complex reactions, orcell-mediated immunity (CMI) reactions (also referred to as delayed-typehypersensitivity (DTH) reactions). (See, e.g., Fundamental Immunology(William E. Paul ed., Raven Press, N.Y., 3rd ed. 1993).)

Specific examples of such immunological diseases include the following:rheumatoid arthritis, psoriatic arthritis, autoimmune demyelinativediseases (e.g., multiple sclerosis, allergic encephalomyelitis),endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus,myasthenia gravis, Grave's disease, glomerulonephritis, autoimmunehepatological disorder, inflammatory bowel disease (e.g., Crohn'sdisease), anaphylaxis, allergic reaction, Sjogren's syndrome, type Idiabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyl), and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, and autoimmune gonadal failure.

Accordingly, the methods described herein encompass treatment ofdisorders of B lymphocytes (e.g., systemic lupus erythematosus,Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes),Th₁-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis,psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave'sdisease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, or graft versus host disease), or Th₂-lymphocytes (e.g.,atopic dermatitis, systemic lupus erythematosus, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, or chronic graft versus host disease). Generally, disordersinvolving dendritic cells involve disorders of Th₁-lymphocytes orTh₂-lymphocytes.

In some embodiments, the immunological disorder is a T cell-mediatedimmunological disorder, such as a T cell disorder in which activated Tcells associated with the disorder express CD70. Anti-CD70 bindingagents (e.g., antibodies or derivatives) can be administered to depletesuch CD70-expressing activated T cells. In a specific embodiment,administration of anti-CD70 antibodies or derivatives can depleteCD70-expressing activated T cells, while resting T cells are notsubstantially depleted by the anti-CD70 or derivative. In this context,“not substantially depleted” means that less than about 60%, or lessthan about 70% or less than about 80% of resting T cells are notdepleted.

The anti-CD70 binding agents (e.g., antibodies and derivatives) are alsouseful for treating or preventing a CD70-expressing cancer. Treatment orprevention of a CD70-expressing cancer, according to the methodsdescribed herein, is achieved by administering to a subject in need ofsuch treatment or prevention an effective amount of the anti-CD70antibody or derivative, whereby the antibody or derivative (i) binds toCD70-expressing cancer cells and (ii) exerts a cytotoxic or cytostaticeffect to deplete or inhibit the proliferation of the CD70-expressingcancer cells. In some embodiments, the cytotoxic, cytostatic, orimmunomodulatory is exerted without conjugation to a cytotoxic,cytostatic, or immunomodulatory agent. In some embodiments, thecytotoxic, cytostatic, or immunomodulatory is exerted by conjugation toa cytotoxic, cytostatic, or immunomodulatory agent.

CD70-expressing cancers that can be treated or prevented by the methodsdescribed herein include, for example, different subtypes ofNon-Hodgkin's Lymphoma (indolent NHLs, follicular NHLs, smalllymphocytic lymphomas, lymphoplasmacytic NHLs, or marginal zone NHLs);Hodgkin's disease (e.g., Reed-Sternberg cells); cancers of the B-celllineage, including, e.g., diffuse large B-cell lymphomas, follicularlymphomas, Burkitt's lymphoma, mantle cell lymphomas, B-cell lymphocyticleukemias (e.g., acute lymphocytic leukemia, chronic lymphocyticleukemia); Epstein Barr Virus positive B cell lymphomas; renal cellcarcinomas (e.g., clear cell and papillary); nasopharyngeal carcinomas;thymic carcinomas; gliomas; glioblastomas; neuroblastomas; astrocytomas;meningiomas; Waldenstrom macroglobulinemia; multiple myelomas; andcolon, stomach, and rectal carcinomas. The cancer can be, for example,newly diagnosed, pre-treated or refractory or relapsed. In someembodiments, a CD70-expressing cancer has at least about 15,000, atleast about 10,000 or at least about 5,000 CD70 molecules/cell.

VIII. PHARMACEUTICAL COMPOSITIONS COMPRISING ANTI-CD70 ANTIBODIES ANDDERIVATIVES AND ADMINISTRATION THEREOF

A composition comprising a CD70 binding agent (e.g., an anti-CD70antibody or derivative) can be administered to a subject having or atrisk of having an immunological disorder or a CD70-expressing cancer.The invention further provides for the use of a CD70 binding agent(e.g., an anti-CD70 antibody or derivative) in the manufacture of amedicament for prevention or treatment of a CD70 expressing cancer orimmunological disorder. The term “subject” as used herein means anymammalian patient to which a CD70-binding agent can be administered,including, e.g., humans and non-human mammals, such as primates,rodents, and dogs. Subjects specifically intended for treatment usingthe methods described herein include humans. The antibodies orderivatives can be administered either alone or in combination withother compositions in the prevention or treatment of the immunologicaldisorder or CD70-expressing cancer.

Various delivery systems are known and can be used to administer theCD70 binding agent. Methods of introduction include but are not limitedto intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The CD70 bindingagent can be administered, for example by infusion or bolus injection(e.g., intravenous or subcutaneous), by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,and the like) and can be administered together with other biologicallyactive agents such as chemotherapeutic agents. Administration can besystemic or local.

In specific embodiments, the CD70 binding agent composition isadministered by injection, by means of a catheter, by means of asuppository, or by means of an implant, the implant being of a porous,non-porous, or gelatinous material, including a membrane, such as asialastic membrane, or a fiber. Typically, when administering thecomposition, materials to which the anti-CD70 binding agent does notabsorb are used.

In other embodiments, the anti-CD70 binding agent is delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref.Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek etal., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymericmaterials can be used. (See Medical Applications of Controlled Release(Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); ControlledDrug Bioavailability, Drug Product Design and Performance (Smolen andBall eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol.Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105.) Other controlled release systems are discussed,for example, in Langer, supra.

A CD70 binding agent (e.g., an anti-CD70 antibody or derivative) can beadministered as pharmaceutical compositions comprising a therapeuticallyeffective amount of the binding agent and one or more pharmaceuticallycompatible ingredients. For example, the pharmaceutical compositiontypically includes one or more pharmaceutical carriers (e.g., sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like). Water is a more typical carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include, for example, starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol, and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oralformulations can include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, etc. Examples of suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin. Such compositions will contain a therapeutically effectiveamount of the protein, typically in purified form, together with asuitable amount of carrier so as to provide the form for properadministration to the patient. The formulations correspond to the modeof administration.

In typical embodiments, the pharmaceutical composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the pharmaceutical can alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the pharmaceutical is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thepharmaceutical is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients can bemixed prior to administration.

Further, the pharmaceutical composition can be provided as apharmaceutical kit comprising (a) a container containing a CD70 bindingagent (e.g., an anti-CD70 antibody or derivative) in lyophilized formand (b) a second container containing a pharmaceutically acceptablediluent (e.g., sterile water) for injection. The pharmaceuticallyacceptable diluent can be used for reconstitution or dilution of thelyophilized anti-CD70 antibody or derivative. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The amount of the CD70 binding agent (e.g., anti-CD70 antibody orderivative) that is effective in the treatment or prevention of animmunological disorder or CD70-expressing cancer can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the stage of immunological disorder orCD70-expressing cancer, and should be decided according to the judgmentof the practitioner and each patient's circumstances. Effective dosesmay be extrapolated from dose-response curves derived from in vitro oranimal model test systems.

For example, toxicity and therapeutic efficacy of the anti-CD70 antibodyor derivative can be determined in cell cultures or experimental animalsby standard pharmaceutical procedures for determining the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. A CD70-binding agent (e.g., an anti-CD70 antibodyor derivative) that exhibits a large therapeutic index is preferred.Where a CD70-binding agent exhibits toxic side effects, a deliverysystem that targets the CD70-binding agent to the site of affectedtissue can be used to minimize potential damage non-CD70-expressingcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofthe CD70 binding agent typically lies within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For a CD70 bindingagent used in the method, the therapeutically effective dose can beestimated initially from cell culture assays. A dose can be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ (i.e., the concentration of the test compoundthat achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma can be measured, for example,by high performance liquid chromatography.

Generally, the dosage of an anti-CD70 antibody or derivativeadministered to a patient with an immunological disorder orCD70-expressing cancer is about 0.1 mg/kg to 100 mg/kg of the subject'sbody weight. More typically, the dosage administered to a subject is 0.1mg/kg to 50 mg/kg of the subject's body weight, even more typically 1mg/kg to 30 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 15 mg/kg, 1 mg/kg to12 mg/kg, 1 mg/kg to 10 mg/kg, or 1 mg/kg to 7.5 mg/kg of the subject'sbody weight. Generally, human antibodies have a longer half-life withinthe human body than antibodies from other species due to the immuneresponse to the foreign proteins. Thus, lower dosages of anti-CD70antibody or derivative comprising humanized or chimeric antibodies andless frequent administration is often possible.

A dose of an anti-CD70 binding agent can be administered, for example,daily, once per week (weekly), twice per week, thrice per week, fourtimes per week, five times per week, biweekly, monthly or otherwise asneeded.

In some embodiments, the dosage of an anti-CD70 binding agentcorresponds to a sub-optimal dosage (i.e., below the EC₅₀ for theanti-CD70 binding agent (e.g., an antibody drug conjugate). For example,the dosage of an anti-CD70 binding agent can comprise a dosage selectedfrom the lowest 25%, lowest 15%, lowest 10% or lowest 5% of thetherapeutic window. As used herein, the term “therapeutic window” refersto the range of dosage of a drug or of its concentration in a bodilysystem that provides safe and effective therapy.

In some embodiments, the dosage of an anti-CD70 binding agent (e.g., anantibody drug conjugate) is from about 0.05 mg/kg to about 1 mg/kg, orabout 0.1 mg/kg to about 0.9 mg/kg, or about 0.15 to about 0.75 mg/kg ofthe subject's body weight. Such a dosage can be administered from 1 toabout 15 times per week. Each dose can be the same or different. Forexample, a dosage of about 0.15 mg/kg of an anti-CD70 binding agent canbe administered from 1 to 10 times per four day, five day, six day orseven day period.

In some embodiments, the pharmaceutical compositions comprising the CD70binding agent can further comprise a therapeutic agent (e.g., anon-conjugated cytotoxic or immunomodulatory agent such as, for example,any of those described herein). The anti-CD70 binding agent also can beco-administered in combination with one or more therapeutic agents forthe treatment or prevention of immunological disorders orCD70-expressing cancers. For example, combination therapy can include atherapeutic agent (e.g., a cytostatic, cytotoxic, or immunomodulatoryagent, such as an unconjugated cytostatic, cytotoxic, orimmunomodulatory agent such as those conventionally used for thetreatment of cancers or immunological disorders). Combination therapycan also include, e.g., administration of an agent that targets areceptor or receptor complex other than CD70 on the surface of activatedlymphocytes, dendritic cells or CD70-expressing cancer cells. An exampleof such an agent includes a second, non-CD70 antibody that binds to amolecule at the surface of an activated lymphocyte, dendritic cell orCD70-expressing cancer cell. Another example includes a ligand thattargets such a receptor or receptor complex. Typically, such an antibodyor ligand binds to a cell surface receptor on activated lymphocytes,dendritic cell or CD70-expressing cancer cell and enhances the cytotoxicor cytostatic effect of the anti-CD70 antibody by delivering acytostatic or cytotoxic signal to the activated lymphocyte, dendriticcell or CD70-expressing cancer cell. Such combinatorial administrationcan have an additive or synergistic effect on disease parameters (e.g.,severity of a symptom, the number of symptoms, or frequency of relapse).

With respect to therapeutic regimens for combinatorial administration,in a specific embodiment, an anti-CD70 binding agent is administeredconcurrently with a therapeutic agent. In another specific embodiment,the therapeutic agent is administered prior or subsequent toadministration of the anti-CD70 antibody or derivative, by at least anhour and up to several months, for example at least an hour, five hours,12 hours, a day, a week, a month, or three months, prior or subsequentto administration of the anti-CD70 antibody or derivative. In someembodiments, the subject is monitored following administration of theanti-CD70 binding agent, and optionally the therapeutic agent.

The therapeutic agent can be, for example, any agent that exerts atherapeutic effect on cancer cells or activated immune cells. Typically,the therapeutic agent is a cytotoxic or immunomodulatory agent. Suchcombinatorial administration can have an additive or synergistic effecton disease parameters (e.g., severity of a symptom, the number ofsymptoms, or frequency of relapse).

Useful classes of cytotoxic or immunomodulatory agents include, forexample, antitubulin agents, auristatins, DNA minor groove binders, DNAreplication inhibitors, alkylating agents (e.g., platinum complexes suchas cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, pre-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, and the like.

Individual cytotoxic or immunomodulatory agents include, for example, anandrogen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine,bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin,carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine,cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B,dacarbazine, dactinomycin (actinomycin), daunorubicin, decarbazine,docetaxel, doxorubicin, an estrogen, 5-fluordeoxyuridine,5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel, plicamycin, procarbizine, rapamycin(Sirolimus), streptozotocin, tenoposide, 6-thioguanine, thioTEPA,topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.

In some typical embodiments, the therapeutic agent is a cytotoxic agent.Suitable cytotoxic agents include, for example, dolastatins (e.g.,auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g.,enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel anddocetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,echinomycin, combretastatin, netropsin, epothilone A and B,estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide,eleutherobin, and mitoxantrone.

In some embodiments, the cytotoxic agent is a conventionalchemotherapeutic such as, for example, doxorubicin, paclitaxel,melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide. Insome embodiments, the therapeutic agent can be a combined therapy, suchas CHOP (Cyclophosphamide, Doxorubicin, Prednisolone and Vincristine),CHOP-R (Cyclophosphamide, Doxorubicin Vincristine, Prednisolone, andrituximab) or ABVD (Doxorubicin, Bleomycin, Vinblastine andDacarbazine). Agents such as CC-1065 analogues, calicheamicin,maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can belinked to the anti-CD70 antibodies or derivatives thereof.

In specific embodiments, the cytotoxic or cytostatic agent is auristatinE (also known in the art as dolastatin-10) or a derivative thereof.Typically, the auristatin E derivative is, e.g., an ester formed betweenauristatin E and a keto acid. For example, auristatin E can be reactedwith paraacetyl benzoic acid or benzoylvaleric acid to produce AEB andAEVB, respectively. Other typical auristatin derivatives include AFP,MMAF, and MMAE. The synthesis and structure of auristatin E and itsderivatives are described in U.S. Patent Application Publication Nos.20030083263 and 20050009751), International Patent Application No.PCT/US03/24209, International Patent Application No. PCT/US02/13435, andU.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860;5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284;5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744;4,879,278; 4,816,444; and 4,486,414.

In specific embodiments, the cytotoxic agent is a DNA minor groovebinding agent. (See, e.g., U.S. Pat. No. 6,130,237.) For example, insome embodiments, the minor groove binding agent is a CBI compound. Inother embodiments, the minor groove binding agent is an enediyne (e.g.,calicheamicin).

Examples of anti-tubulin agents include, but are not limited to, taxanes(e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vincaalkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine),and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Otherantitubulin agents include, for example, baccatin derivatives, taxaneanalogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid,estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins,discodermolide, and eleutherobin.

In some embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari etal., 1992, Cancer Res. 52:127-131).

In some embodiments, the therapeutic agent is not a radioisotope. Insome embodiments, the therapeutic agent is not ricin or saporin.

In certain embodiments, the therapeutic agent is an anti-VEGF agent,such as AVASTIN (bevacizumab) or NEXAVAR (Sorafenib); a PDGF blocker,such as SUTENT (sunitinib malate); or a kinase inhibitor, such asNEXAVAR (sorafenib tosylateor).

In some embodiments, the cytotoxic or immunomodulatory agent is anantimetabolite. The antimetabolite can be, for example, a purineantagonist (e.g., azothioprine or mycophenolate mofetil), adihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir,gangcyclovir, zidovudine, vidarabine, ribavarin, azidothymidine,cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine,poscarnet, or trifluridine.

In other embodiments, the cytotoxic or immunomodulatory agent istacrolimus, cyclosporine or rapamycin. In further embodiments, thecytoxic agent is aldesleukin, alemtuzumab, alitretinoin, allopurinol,altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene,bexarotene, calusterone, capecitabine, celecoxib, cladribine,Darbepoetin alfa, Denileukin diftitox, dexrazoxane, dromostanolonepropionate, epirubicin, Epoetin alfa, estramustine, exemestane,Filgrastim, floxuridine, fludarabine, fulvestrant, gemcitabine,gemtuzumab ozogamicin, goserelin, idarubicin, ifosfamide, imatinibmesylate, Interferon alfa-2a, irinotecan, letrozole, leucovorin,levamisole, meclorethamine or nitrogen mustard, megestrol, mesna,methotrexate, methoxsalen, mitomycin C, mitotane, nandrolonephenpropionate, oprelvekin, oxaliplatin, pamidronate, pegademase,pegaspargase, pegfilgrastim, pentostatin, pipobroman, plicamycin,porfimer sodium, procarbazine, quinacrine, rasburicase, Sargramostim,streptozocin, tamoxifen, temozolomide, teniposide, testolactone,thioguanine, toremifene, Tositumomab, Trastuzumab, tretinoin, uracilmustard, vairubicin, vinblastine, vincristine, vinorelbine orzoledronate.

In additional embodiments, the therapeutic agent is an antibody, such asa humanized anti HER2 monoclonal antibody, RITUXAN (rituximab;Genentech; a chimeric anti CD20 monoclonal antibody); OVAREX (AltaRexCorporation, MA); PANOREX (Glaxo Wellcome, NC; a murine IgG2a antibody);Cetuximab Erbitux (Imclone Systems Inc., NY; an anti-EGFR IgG chimericantibody); Vitaxin (MedImmune, Inc., MD; Campath I/H (Leukosite, MA; ahumanized IgG1 antibody); Smart MI95 (Protein Design Labs, Inc., CA; ahumanized anti-CD33 IgG antibody); LymphoCide (Immunomedics, Inc., NJ; ahumanized anti-CD22 IgG antibody); Smart ID10 (Protein Design Labs,Inc., CA; a humanized anti-HLA-DR antibody); Oncolym (Techniclone, Inc.,CA; a radiolabeled murine anti-HLA-Dr10 antibody); Allomune(BioTransplant, CA; a humanized anti-CD2 mAb); Avastin (Genentech, Inc.,CA; an anti-VEGF humanized antibody); Epratuzamab (Immunomedics, Inc.,NJ and Amgen, CA; an anti-CD22 antibody); CEAcide (Immunomedics, NJ; ahumanized anti-CEA antibody); or an anti-CD40 antibody (e.g., asdisclosed in U.S. Pat. No. 6,838,261).

Other suitable antibodies include, but are not limited to, antibodiesagainst the following antigens: CA125, CA15-3, CA19-9, L6, Lewis Y,Lewis X, alpha fetoprotein, CA 242, placental alkaline phosphatase,prostate specific membrane antigen, prostatic acid phosphatase,epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4,anti-transferrin receptor, p97, MUC1-KLH, CEA, gp100, MART1, ProstateSpecific Antigen, IL-2 receptor, CD20, CD52, CD30, CD33, CD22, humanchorionic gonadotropin, CD38, CD40, mucin, P21, MPG, and Neu oncogeneproduct.

In some embodiments, the therapeutic agent is an immunomodulatory agent.The immunomodulatory agent can be, for example, gancyclovir, etanercept,tacrolimus, cyclosporine, rapamycin, REVLIMID (lenalidomide),cyclophosphamide, azathioprine, mycophenolate mofetil or methotrexate.Alternatively, the immunomodulatory agent can be, for example, aglucocorticoid (e.g., cortisol or aldosterone) or a glucocorticoidanalogue (e.g., prednisone or dexamethasone).

In some typical embodiments, the immunomodulatory agent is ananti-inflammatory agent, such as arylcarboxylic derivatives,pyrazole-containing derivatives, oxicam derivatives and nicotinic acidderivatives. Classes of anti-inflammatory agents include, for example,cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and leukotrienereceptor antagonists. In some embodiments, the immodulatory agent is acytokine, such as G-CSF, GM-CSF or IL-2.

Suitable cyclooxygenase inhibitors include meclofenamic acid, mefenamicacid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen,ibuprofen, indomethacin, ketoprofen, nabumetone, naproxen, sulindac,tenoxicam, tolmetin, and acetylsalicylic acid.

Suitable lipoxygenase inhibitors include redox inhibitors (e.g.,catechol butane derivatives, nordihydroguaiaretic acid (NDGA),masoprocol, phenidone, Ianopalen, indazolinones, naphazatrom,benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g.,hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivativesthereof, methoxytetrahydropyran, boswellic acids and acetylatedderivatives of boswellic acids, and quinolinemethoxyphenylacetic acidssubstituted with cycloalkyl radicals), and precursors of redoxinhibitors.

Other suitable lipoxygenase inhibitors include antioxidants (e.g.,phenols, propyl gallate, flavonoids and/or naturally occurringsubstrates containing flavonoids, hydroxylated derivatives of theflavones, flavonol, dihydroquercetin, luteolin, galangin, orobol,derivatives of chalcone, 4,2′,4′-trihydroxychalcone, ortho-aminophenols,N-hydroxyureas, benzofuranols, ebselen and species that increase theactivity of the reducing selenoenzymes), iron chelating agents (e.g.,hydroxamic acids and derivatives thereof, N-hydroxyureas,2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acidand 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containingcompounds (e.g., ketoconazole and itraconazole), phenothiazines, andbenzopyran derivatives.

Yet other suitable lipoxygenase inhibitors include inhibitors ofeicosanoids (e.g., octadecatetraenoic, eicosatetraenoic,docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and estersthereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol,15-monohydroxyeicosatetraenoic, 15-monohydroxy-eicosatrienoic and15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5),compounds interfering with calcium flows, phenothiazines,diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid,caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA),hydroxyphenylretinamide, Ionapalen, esculin, diethylcarbamazine,phenantroline, baicalein, proxicromil, thioethers, diallyl sulfide anddi-(1-propenyl)sulfide.

Leukotriene receptor antagonists include calcitriol, ontazolast, BayerBay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark ETH-615, LillyLY-293111, Ono ONO-4057, Terumo TMK-688, Boehringer Ingleheim BI-RM-270,Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono ONO LB457, Pfizer105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP 66153,SmithKline Beecham SB-201146, SmithKline Beecham SB-201993, SmithKlineBeecham SB-209247, Searle SC-53228, Sumitamo SM 15178, American HomeProducts Way 121006, Bayer Bay-o-8276, Warner-Lambert CI-987,Warner-Lambert CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283,MacroNex MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, OnoONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893,Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698, ShionoogiS-2474, Searle SC-41930, Searle SC-50505, Searle SC-51146, SearleSC-52798, SmithKline Beecham SKand F-104493, Leo Denmark SR-2566, TanabeT-757 and Teijin TEI-1338.

The invention is further described in the following examples, which arein not intended to limit the scope of the invention. Cell linesdescribed in the following examples were maintained in culture accordingto the conditions specified by the American Type Culture Collection(ATCC) or Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,Braunschweig, Germany (DMSZ), or as otherwise known. Cell culturereagents were obtained from Invitrogen Corp., Carlsbad, Calif.

Example 1 Production of Humanized Anti-CD70 Antibody Variants

The nucleotide and amino acid sequences of the heavy and light variableregions of antiCD70 murine monoclonal antibody, 1F6, and a chimericvariant of 1F6, c1F6, are set forth as SEQ ID NOS:1, 2, 21 and 22,respectively. (See also U.S. Patent Application No. 60/645,355, filedJan. 19, 2005). Human acceptor sequences for humanization of c1F6 werechosen from human germline exon V_(H), J_(H), Vκ and Jκ sequences.Acceptor sequences for c1F6 V_(H) domain humanization were chosen fromgermline V_(H) exons V_(H)1-18 (Matsuda et al., 1993, Nature Genetics3:88-94) or V_(H)1-2 (Shin et al., 1991, EMBO J. 10:3641-3645) and J_(H)exon J_(H)-6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582).Germline Vκ exon B3 (Cox et al., 1994, Eur. J. Immunol. 24:827-836) andJκ exon Jκ-1 (Hieter et al., 1982, J. Biol. Chem. 257:1516-1522) werechosen as acceptor sequences for c1F6 V_(L) domain humanization. 1F6murine CDRs, determined according to the Kabat definition, were graftedonto the chosen human germline template. Briefly, synthetic overlappingoligonucleotides spanning the humanized V_(H) or V_(L) domain weregenerated and PCR overlap extension was used to assemble each domain.Restriction sites incorporated into the PCR product were used todirectionally clone the V_(H) and V_(L) domain into a pCMV expressionvector in frame with human IgG1 constant domains or Kappa constantdomain, respectively.

Several framework positions were chosen for reintroduction of mousedonor residues. These were positions H46, H67, H68, H69, H70, H71, H80,H81, H82, H82A and H91 in the V_(H) domain, according to the Kabatnumbering convention. No framework positions were altered in the V_(L)domain, although mouse CDR1 residues at positions L25 and L33 werechosen for introduction of the human acceptor residue for that position.

Several variants of humanized 1F6 were generated by incorporatingdifferent combinations of mouse framework donor residues in the V_(H)domain or human CDR residues in the V_(L) domain. These variants aresummarized in Tables 2 and 3 below.

TABLE 2 V_(H) VH exon acceptor variant sequence donor framework residueshV_(H) A VH1-18 H71, H91 hV_(H) B VH1-18 H71 hV_(H) C VH1-18 H91 hV_(H)D VH1-18 none hV_(H) E VH1-2 none hV_(H) F VH1-18 H67, H68, H69, H70,H71 hV_(H) G VH1-18 H80, H81, H82, H82A hV_(H) H VH1-18 H67, H68, H69,H70, H71, H80, H81, H82, H82A hV_(H) I VH1-18 H46, H71, H91 hV_(H) JVH1-2 H46 hV_(H) K VH1-2 H71 hV_(H) L VH1-2 H46, H71 hV_(H) M VH1-18H46, H67, H68, H69, H70, H71 hV_(H) N VH1-18 H69, H70, H71, H80

TABLE 3 V_(L) Acceptor variant CDR residue hV_(L) A none hV_(L) B L25hV_(L) C L33 hV_(L) D L25, L33

The differences between some of the humanized variants with the murineand human V_(H) sequences are illustrated in FIGS. 1 and 2. An alignmentof humanized 1F6 V_(H) variants hV_(H)E and hV_(H)J with 1F6 mV_(H) andhuman germline V_(H) exon V_(H)1-2 and J_(H) exon JH6 is shown inFIG. 1. An alignment of humanized 1F6 V_(H) variants hV_(H)H and hV_(H)Mwith 1F6 mV_(H) and human germline V_(H) exon VH1-18 and J_(H) exon JH6is shown in FIG. 2. An alignment of humanized 1F6 V_(L) variant hV_(L)Awith 1F6 mV_(L) and human germline V_(κ) exon B3 and J_(κ) exon Jκ-1 isshown in FIG. 3.

Example 2 Binding Affinities of Humanized 1F6 Variants

Humanized 1F6 variants HDLA (hV_(H)D and hV_(L)A), HHLA (hV_(H)H andhV_(L)A), and HJLA (hV_(H)J and hV_(L)A), were selected for bindingaffinity analysis. One mg of each humanized antibody and c1F6 weretransiently expressed in 293 cells and labeled with europium using theEu-N1 iodoacetamido chelate (Perkin Elmer). Saturation binding to apanel of CD70 positive cell lines was assessed for each labeledantibody. The cell lines selected were ACHN, Caki-2, Caki-1, and 786-Owith antigen copies/cell determined by quantitative flow cytometry (orfluorescence activated cell sorting, i.e., FACS) of 30,000, 99,000,235,000, and 252,000 respectively.

Europium-labeled antibodies were incubated with cells for 1 hour at 4°C. over a range of concentrations in 96 well plates. Followingincubation europium was released by resuspension of the cells inEnhancement Buffer (Perkin Elmer). Fluorescence was read in a Fusion HTplate reader using a top detector format and excitation of 335 nm andemission of 620 nm Data was fit to a one binding site hyperbola usingGraphPad Prism 4. The results are shown below in Table 4.

TABLE 4 Antigen/ Apparent binding affinity K_(D) (nM) Cell line cellc1F6 h1F6 HDLA h1F6 HHLA h1F6 HJLA ACHN 30,000 0.30 1.44 0.29 0.68Caki-1 235,000 1.28 1.29 1.22 1.36 Caki-2 99,000 0.26 0.86 0.15 0.37786-O 252,000 0.56 0.55 0.28 0.46

The K_(D) values for the humanized variants are very similar to c1F6 onall of the cell lines tested, confirming that the humanization processdid not significantly reduce antigen binding activity.

Example 3 ADCC Activity of Humanized 1F6

The ability of humanized 1F6 antibody variants to mediate ADCC againstthe CD70⁺ cell lines WIL2-S, 786-O and 769-P was measured using astandard ⁵¹Cr release assay. The HHLA, HJLA and HELA variants ofhumanized 1F6 lysed WIL-2S target cells equivalently and in a dosedependent manner. In contrast, tumor cells treated with CD70-bindingmurine 1F6 (m1F6) or non-binding control human Ig (hIg) were not killed(FIG. 4A). Similarly, humanized 1F6 mediated the lysis of two renal cellcarcinoma targets in a manner comparable to chimeric 1F6 (FIG. 4B).

Example 4 CDC Activity of Humanized 1F6

The ability of humanized 1F6 to mediate CDC was examined using amultiple myeloma cell line (LP-1) and two lymphoma cell lines (MHHPreB-1 and WIL2-S). Target cells were treated with graded doses ofchimeric 1F6, humanized 1F6 HJLA or a non-binding human Ig control inthe presence of normal human serum. After incubation at 37° C. for 2hours, lysed cells were identified by flow cytometry after the additionof propidium iodide (5 μg/mL). Cells stained with propidium iodide wereconsidered to have lost plasma membrane integrity as a result ofantibody-mediated complement activation and formation of the membraneattack complex. Using this assay, chimeric 1F6 and humanized 1F6mediated dose-dependent lysis of each target in an equivalent manner(FIG. 5).

Example 5 ADCP Activity of Humanized 1F6

The ability of humanized 1F6 to mediate phagocytosis was examined usingthe CD70⁺ renal cell carcinoma line 786-O pre-labeled with a redfluorescent membrane dye. Target cells were treated with graded doses ofchimeric 1F6, humanized 1F6 HJLA or a non-binding human Ig control andwere then mixed with macrophages generated from adherent peripheralblood monocytes cultured in GM-CSF. After incubation at 37° C. for 1hour, the macrophages were detected with a green fluorescent antibody tothe macrophage cell surface marker CD11b. Macrophages that hadphagocytosed tumor cells were identified by dual red and greenfluorescence as detected by flow cytometry. The presence of tumor cellswithin macrophages in the dual-positive population was confirmed byfluorescence microscopy. As shown in FIG. 6, chimeric and humanized 1F6facilitated phagocytosis of target cells in an antibody-dose dependentfashion and to an equivalent degree. In contrast, target cells incubatedwith a non-binding control antibody were minimally engulfed bymacrophages.

Example 6 In Vitro Cytotoxicity Activity of Humanized 1F6 Variant DrugConjugates

Humanized 1F6 variants HELA (hV_(H)E and hV_(L)A), HHLA, HJLA, and HMLA(hV_(H)M and hV_(L)A), and c1F6 were transiently expressed in 293 cellsand conjugated to vcMMAF (described in U.S. Ser. No. 10/983,340;published as U.S. Patent Publication No. 2005-0238649, Oct. 27, 2005) ata loading level of an average of eight drug units per antibody. Theresulting conjugates, h1F6 HELA-F8, h1F6 HHLA-F8, h1F6 HJLA-F8, h1F6HMLA-F8, and c1F6-F8 were tested for cytotoxicity against two CD70expressing cell lines, 786-O and Caki-1. The conjugates were incubatedwith the cells for 92 hours, followed by addition of 50 μM resazurin.After a 4 hour incubation period, dye reduction was measured using aFusion HT fluorescent plate reader (Packard Instruments, Meriden,Conn.). The results of triplicate sampling are shown below in Table 5.The IC₅₀ values of all four humanized variants are active withintwo-fold of c1F6 on both cell lines tested with a potency ranking ofc1F6-F8>h1F6 HHLA-F8>h1F6 HMLA-F8>h1F6 HJLA-F8>h1F6 HELA-F8.

TABLE 5 No. of h1F6- mouse FR Caki-1 786-O vcMMAF residues IC50 [ng/ml]IC50 [ng/ml] h1F6 HELA- 0 3.4 5.2 F8 (mean = 2.87, n = 3) (mean = 3.9, n= 3) h1F6 9 1.4 2.3 HHLA-F8 (mean = 1.87, n = 3) (mean = 1.93, n = 3)h1F6 HJLA- 1 2.2 3.4 F8 (mean = 2.3, n = 3) (mean = 3.03, n = 3) h1F6 61.8 2.8 HMLA-F8 (mean = 2.07, n = 3) (mean = 2.03, n = 3) c1F6(293)- 01.8 2.4 F8 (mean = 2.17, n = 3) (mean = 1.45, n = 3)

Example 7 In Vivo Screening of Humanized 1F6 Drug Conjugates

Humanized 1F6 variants HDLA, HHLA, HJLA, and HELA, were transientlyexpressed in 293 cells and conjugated to mcMMAF (described in U.S. Ser.No. 10/983,340; published as U.S. Patent Publication No. 2005-0238649,Oct. 27, 2005) at a loading level of eight drug units per antibody. Anefficacy study of a single dose at 3 mg/kg or 10 mg/kg was performed ina 786-O renal cell carcinoma solid tumor model in nude mice. Tumorvolume was measured regularly for 80 days post-tumor implant. Theresults indicate that tumor volume was greatly reduced in all treatedmice in comparison to untreated mice, and all humanized 1F6 variantsconjugated to mcMMAF were comparable in efficacy to c1F6mcMMAF.

Example 8 In Vivo Activity of Humanized 1F6 in SCID Mouse XenograftModels of Disseminated Lymphoma and Multiple Myeloma

The in vivo antitumor activity of humanized 1F6 (HJLA) was examined indisseminated lymphoma and multiple myeloma xenograft mouse models. Toestablish disseminated disease, 1×10⁶ Raji or 1×10⁷ MM1.S or L363 cellswere injected into the lateral tail vein of C.B.-17 SCID mice. Mice weredosed with humanized 1F6 (HJLA) or control non-binding antibody byintraperitoneal (i.p.) injection every four days for a total of sixdoses (Raji) or by intravenous injection into the lateral tail vein onceweekly for a total of four weeks (MM.1S and L363) starting one day aftercell implant. Disease requiring euthanasia was manifested by hunchedposture and lack of grooming, weight loss, cranial swelling and hindlimb paralysis, or, in L363-bearing mice, the development of palpablelymphoid tissue-associated tumors.

The results show that, in each tumor model (FIGS. 7A, 7B and 7C),survival of mice treated with humanized 1F6 was significantly prolongedcompared to that of untreated mice or mice receiving non-binding controlantibody. The effect of humanized 1F6 treatment was further evaluated inmultiple myeloma xenografts (L363 and MM.1S cells) by measuring thelevel of tumor-derived monoclonal protein (λ light chain) in the sera ofindividual mice. As shown in FIGS. 7B and 7C (right panels), circulatingλ light chain concentrations were significantly lower in mice treatedwith humanized 1F6 as compared to untreated mice. Mean serum levels of λlight chain in L363-bearing mice treated with humanized 1F6 were 0.006μg/mL compared to 0.10 μg/mL in sera of untreated mice. Similarly, λlight chain levels in humanized 1F6-treated MM.1S-bearing mice were 0.03μg/mL compared to 1.25 μg/mL in untreated mice. These results wereconsistent with the increased survival rates of the mice (FIGS. 7B and7C, right panels).

Example 9 In Vitro Deletion of CD70⁺ Antigen-Specific T Cells byHumanized 1F6 Antibody

To test the ability of humanized 1F6 antibody to depleteantigen-specific activated T cells, PBMC from a normal donor expressingHLA-A0201 were stimulated with the M1 peptide in the presence or absenceof varying concentrations of humanized anti-CD70 antibody. Humanized 1F6antibody (HJLA) was prepared as described above. PBMC were seeded in a24-well plate at a concentration of 0.5×10⁶ cells/ml with 5 μg/ml M1peptide in 2 ml of medium supplemented with IL-2 and IL-15. On day 5,half of the culture supernatant was replaced with freshcytokine-containing medium. On day 9, the percentage of antigen-reactivecells (the CD8⁺/Vβ17⁺ population) was determined by flow cytometricanalysis of cells stained with FITC-conjugated anti-Vβ17- andPE-Cy5-conjugated anti-CD8 antibodies.

FIG. 8A shows that antigen-specific CD8⁺/Vβ17⁺ cells expanded tocomprise 33% of all viable cells within the culture in the absence ofantibody. In contrast, addition of humanized 1F6 to the cultures on day0 significantly limited expansion of the antigen-reactive population inan antibody-dose dependent manner. These results show that humanized 1F6selectively targets and prevents the expansion of antigen-activated Tcells.

In a second study (FIG. 8B), M1-peptide stimulated cultures wereuntreated or treated with humanized 1F6 in the absence or presence ofantibody that specifically blocks FcγRIII (CD16). In untreated cultures,the antigen-specific CD8+Vβ17⁺ population expanded to comprise 39% ofall viable cells within the culture. Addition of humanized 1F6significantly diminished expansion of the reactive population. Thisactivity was largely reversed when FcγRIII receptors were blocked withanti-CD16 specific antibody, indicating that deletion ofpeptide-reactive cells was mediated via humanized 1F6 interaction withFcgRIII-bearing effector cells.

Example 10 Anti-CD70 Antibody does not Affect Antigen-Negative BystanderCells

To determine the effect of 1F6-mediated depletion on antigen-negativebystander T cells, the TCR Vβ family representation of CD4 and CD8lymphocytes was examined in M1-activated cultures that were untreated ortreated with a chimeric variant of 1F6 (c1F6) (human IgG1 isotype) andcompared to resting, non-antigen stimulated PBMC. Chimeric and humanized1F6 variants are comparable in binding affinity, capacity to mediateeffector functions, and ability to deplete activated CD8+ T cellsubsets.

As shown in FIG. 9, stimulation of HLA-A0201⁺ PBMC with M1 peptidecaused the expansion of CD8⁺ cells bearing the Vβ17 TCR approximately30-fold, whereas all other Vβ TCR families tested in CD8⁺ cells and allfamilies tested in the CD4 cell population demonstrated minimal change.In the control population, cell expansion was limited to the Vβ17⁺CD8⁺ Tcell subset, which increased from <1% of CD8⁺ cells to 27%; thisobservation confirms the specificity of the M1-peptide immune response.Unlike T cells stimulated in the absence of CD 70-specific antibody,expansion of M1-peptide specific CD8⁺ cells was prevented by theaddition of c1F6 antibody to the culture. In the presence of c1F6antibody, the percent Vβ17⁺CD8⁺ cells was comparable to that of resting,non-peptide stimulated cells. Treatment with c1F6 antibody did notsignificantly perturb the relative representations of other CD8⁺ or CD4⁺Vβ TCR families; no group was observed to be eliminated. These datademonstrate that exposure to c1F6 antibody selectively depletes CD70⁺activated T cells without causing detectable collateral damage tobystander T cell populations.

Example 11 Mouse Xenograft Model of Renal Cell Carcinoma

A 786-O subcutaneous xenograft model was used to evaluate antitumoractivity of anti-CD70 ADCs administered at different dosages andschedules. Subcutaneous 786-O tumors were initiated in nude mice byimplanting tumor fragments (N=5 or 6/group) of approximated 30 mm³.Tumor growth was allowed to establish and treatment began when averagetumor size was approximately 100 mm³. Tumor dimensions were determinedby caliper measurements to monitor growth. Tumor size was calculatedusing the formula of (length×width²)/2. In the absence of any treatment,mean tumor volume increased to approximately 600 mm³ within 40 to 50days after tumor implantation (see FIG. 10A). A dose-dependent effect intumor growth suppression was observed in mice which received eitherhumanized 1F6-mcMMAF4 (HJLA with a loading level of an average of fourdrug units per antibody) or humanized 1F6-vcMMAF4 (HJLA with a loadinglevel of an average of four drug units per antibody). Detectable delayin tumor growth was observed even at 0.5 and 0.17 mg/kg of h1F6-mcMMAF4and h1F6-vcMMAF4, respectively.

Tumor growth was also assessed by time needed for tumors to quadruple insize (see FIG. 10B). Treatment with either h1F6-mcMMAF4 or h1F6-vcMMAF4at 0.17 mg/kg significantly delayed the growth of tumors. This delay wasobserved when the ADCs were given on a q4d×4 or q4d×10 schedule.However, additional administrations as exemplified by the q4d×10schedule appeared to have a stronger growth inhibitory activity comparedto the q4d×4 schedule.

Example 12 Expression of CD70 on Multiple Myeloma Cell Lines

Cell surface CD70 expression was evaluated in a panel of multiplemyeloma cell lines (Table 6). Copy number of CD70 molecules expressed byeach cell line was determined by quantitative flow cytometry using theQIFIKit® (Dako, Carpinteria, Calif.). Response of these cells toanti-CD70 ADC-mediated cytotoxicity was determined. In this model, theactivity of chimeric anti-CD70 ADCs is a proxy for activity of humananti-CD70 ADCs. Both chimeric 1F6(c1F6)-vcMMAF4 and c1F6-mcMMAF4 werecytotoxic against CD70-expressing multiple myeloma cells. The IC₅₀values obtained with c1F6-vcMMAF4 ranged from 1.2-160 ng/mL while thatobtained with c1F6-mcMMAF4 ranged from 1.7-500 ng/mL.

TABLE 6 Cytotoxic Activity of Chimeric Anti-CD70 ADCs against MultipleMyeloma Cell Lines IC₅₀ (ng/mL) CD70 copies/cell c1F6-vcMMAF4c1F6-mcMMAF4 MM.1S 14,000 20 22 MM.1R 25,000 13 20 AMO-1 92,000 16 38JJN-3 19,000 46 61 L363 13,000 78 210 LB 45,000 80 500 U266 155,000 1.21.7 LP-1 34,000 160 155 MOLP-8 9,000 73 33

Example 13 Mouse Xenograft Models of Multiple Myeloma

The in vivo activity of anti-CD70 ADCs in xenograft models of multiplemyeloma was further examined. Human multiple myeloma cell lines MM-1S(FIGS. 11A & 11B) or L363 (FIGS. 12A & 12B) were resuspended inRPMI-1640 medium at the concentration of 10×10⁶ cells/300 μL. Toestablish tumors 300 μL of cell suspension were injected intravenouslythrough the tail veins of SCID mice. In the MM-1S model, untreated micesuccumbed to the injected tumor cells and manifested symptoms around 40days post tumor implant including hind limb paralysis, hunched posture,cranial swelling, and/or scruffy coat. Mice were euthanized when theydemonstrated one or more of these symptoms. Both h1F6(HJLA)-vcMMAF4 andh1F6(HJLA)-mcMMAF4 provided significant survival benefits to tumorbearing mice compared to control non-binding IgG-vcMMAF4 and IgG-mcMMAF4(see FIG. 11A). Tumor burden in the MM-1S model was also assessed byenumerating the number of bone marrow cells expressing human CD138, aplasma cell marker expressed by the MM-1S cells. Bone marrow cells wererecovered from mice that were euthanized due to manifestation of symptomor at the end of the experiment on day 122, and the number ofCD138-expressing MM-1S cells was determined by flow cytometry. Comparedto untreated mice, both control IgG-vcMMAF4 and IgG-mcMMAF4 did notsignificantly reduce the number of CD138-expressing cells in the bonemarrow. On the other hand, h1F6-vcMMAF4 and h1F6-mcMMAF4 significantlyreduce tumor burden as demonstrated by much lower number of bone marrowCD138-expressing cells compared to the control ADCs (see FIG. 11B).

In the L363 model, disseminated tumor masses develop at multiplelocations in mice receiving no treatment, and tumor masses becamepalpable around 40 days after tumor injection, at which tumor bearingmice would be euthanized. Similar to the MM-1S model, controlIgG-vcMMAF4 provided no survival advantage, whereas h1F6-vcMMAF4significantly prolonged survival (see FIG. 12A). Since L363 cellssecrete immunoglobulin lambda light chain (λ LC), tumor burden can bedetermined by monitoring the level of human λ LC in the plasma of tumorbearing mice. An ELISA was used to detect secreted λ LC. Ninety six-wellflat-bottom Immuno plates (Nunc Maxisorp, #442404, Nalge NuncInternational, Rochester, N.Y.) was coated with 100 μL/well of goatanti-human Ig (Southern Biotech #2010-01, Birmingham, Ala.) at 2 μg/mLin 0.1M sodium carbonate/bicarbonate overnight at 4° C. Wells werewashed 5× with 1×PBST (PBS, 0.05% Tween-20), and blocked with 200μL/well of 1% BSA/PBST (0.05% Tween-20) for 1 hour at room temperature.After 5 washes with 1×PBST, serially diluted human λ LC-containing mouseserum samples were added. Purified human λ LC (Bethyl labs, #P80-127,Montgomery, Tex.) was used as the standard. After one hour of incubationat room temperature, wells were washed 5 times with 1×PBST. HRP-goatanti-human lambda chain specific F(ab′)₂ (Southern Biotech #2072-05) at1:4000 dilution in 1% BSA/PBST was added. After an additional one hourincubation at room temperature, wells were washed 5 times with 1×PBST.TMB substrate 100 μL/well (Sigma, #T8665, St. Louis, Mo.) was used todetect captured λ LC. FIG. 12B shows the results at forty days afterL363 cell implant serum. λ LC levels were comparable between theuntreated mice and the IgG-vcMMAF4-treated mice. In contrast, serum λ LClevels in the h1F6-vcMMAF4-treated mice were significantly lower,confirming the ability of anti-CD70 ADC to reduce tumor burden in micebearing multiple myeloma xenografts.

Example 14 Expression of CD70 on Hodgkin's and Glioblastoma Cell Lines

Cell surface CD70 expression was also evaluated in panels of Hodgkin'sdisease (Table 7) and glioblastoma cell lines (Table 8). The copy numberof CD70 molecules expressed by each cell line was determined byquantitative flow cytometry using the QIFIKit® (Dako, Carpinteria,Calif.). The response of these cells to chimeric anti-CD70 ADC-mediatedcytotoxicity was determined. In this model, the activity of chimericanti-CD70 ADCs is a proxy for activity of human anti-CD70 ADCs. Bothchimeric 1F6(c1F6)-vcMMAF4 and c1F6-mcMMAF4 were cytotoxic against theseCD70-expressing cell lines. In the Hodgkin's disease panel, the IC₅₀values obtained with c1F6-vcMMAF4 ranged from 0.41-42 ng/mL while thatobtained with c1F6-mcMMAF4 ranged from 5.2-310 ng/mL (Table 7). In theglioblastoma panel, the IC₅₀ values obtained with h1F6-vcMMAF4 rangedfrom 2.3-27 ng/mL while that obtained with h1F6-mcMMAF4 ranged from15-110 ng/mL (Table 8).

TABLE 7 Cytotoxic Activity of Anti-CD70 ADCs against Hodgkin's DiseaseCell Lines IC₅₀ (ng/mL) CD70 copies/cell c1F6-vcMMAF4 c1F6-mcMMAF4RPMI-6666 21,000 42 230 Hs445 64,000 7.3 310 L428 105,000 1.4 35 KMH2160,000 0.41 5.2 SUP-HD-1 221,000 6.3 53

TABLE 8 Cytotoxic Activity of Chimeric Anti-CD70 ADCs againstGlioblastoma Cell Lines IC₅₀ (ng/mL) CD70 copies/cell h1F6-vcMMAF4h1F6-mcMMAF4 U251 117,000 5.3 15 SNB-19 90,000 12 27 U373MG 70,000 16 35GMS-10 64,000 27 110 DBTRG-05MG 59,000 2.3 20

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various references, including patent applications, patents, andscientific publications, are cited herein, the disclosures of each ofwhich is incorporated herein by reference in its entirety.

What is claimed is:
 1. An isolated polynucleotide encoding a humanizedheavy chain variable region comprising the three CDRs from SEQ ID NO:2and a variable region framework sequence of human germline V_(H)1-2 orV_(H)1-18 and exon J_(H)-6, provided that any of positions H46, H67,H68, H69, H70, H71, H80, H81, H82, H82A and H91 (Kabat numbering) can beoccupied by the amino acid occupying the corresponding position from SEQID NO:2.
 2. The isolated polynucleotide of claim 1, further encoding ahuman IgG constant region.
 3. The isolated polynucleotide of claim 2,wherein the IgG constant region is IgG1 or IgG3.
 4. The isolatedpolynucleotide of claim 2, wherein the humanized heavy chain variableregion and the human IgG constant region together comprise the aminoacid sequence corresponding to residues 20-467 of SEQ ID NO:16 orresidues 20-467 of SEQ ID NO:20.
 5. The isolated polynucleotide of claim4, comprising the nucleotide sequence of nucleotides 58-1401 of SEQ IDNO:15 or nucleotides 58-1401 of SEQ ID NO:19.
 6. The isolatedpolynucleotide of claim 1, wherein position H46 is occupied by the aminoacid occupying the corresponding position from SEQ ID NO:2.
 7. Theisolated polynucleotide of claim 1, wherein at least one of positionsH46, H67, H68, H69, H70, H71, H80, H81, H82, H82A and H91 (Kabatnumbering) in the humanized heavy chain variable region is occupied bythe residue occupying the corresponding position in SEQ ID NO:2.
 8. Theisolated polynucleotide of claim 7, wherein at least one of positionsH71 and H91 (Kabat numbering) in the humanized heavy chain variableregion is occupied by the residue occupying the corresponding positionin SEQ ID NO:2.
 9. The isolated polynucleotide of claim 1, wherein thehumanized heavy chain variable region comprises the amino acid sequenceset forth in SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, oramino acids 20-137 of SEQ ID NO:4.
 10. An isolated polynucleotide,comprising the nucleotide sequence set forth in SEQ ID NO:5, SEQ IDNO:9, SEQ ID NO:13, SEQ ID NO:17, or nucleotides 58-411 of SEQ ID NO:3.11. An isolated polynucleotide encoding a humanized light chain variableregion comprising the three CDRs from SEQ ID NO:22 and a variable regionframework sequence of human germline Vκ exon B3 and Jκ exon Jκ−1,provided that any of positions L25 and L33 can be occupied by the aminoacid occupying the corresponding position from SEQ ID NO:22.
 12. Theisolated polynucleotide of claim 11, further encoding a light chainconstant region.
 13. The isolated polynucleotide of claim 12, whereinthe light chain constant region is a human kappa constant domain. 14.The isolated polynucleotide of claim 12, wherein the humanized lightchain variable region and the light chain constant region togethercomprise the amino acid sequence corresponding to residues 21-238 of SEQID NO:26.
 15. The isolated polynucleotide of claim 14, comprising thenucleotide sequence of nucleotides 61-714 of SEQ ID NO:25.
 16. Theisolated polynucleotide of claim 11, wherein at least one of positionsL25 and L33 in the humanized light chain variable region is occupied bythe residue occupying the corresponding position in SEQ ID NO:22. 17.The isolated polynucleotide of claim 11, wherein the humanized lightchain variable region comprises the amino acid sequence of SEQ ID NO:24.18. An isolated polynucleotide, comprising the nucleotide sequence setforth in SEQ ID NO:23.
 19. The isolated polynucleotide of claim 11,further encoding a humanized heavy chain variable region comprising thethree CDRs from SEQ ID NO:2 and a variable region framework sequence ofhuman germline V_(H)1-2 or V_(H)1-18 and exon J_(H)-6, provided that anyof positions H46, H67, H68, H69, H70, H71, H80, H81, H82, H82A and H91(Kabat numbering) can be occupied by the amino acid occupying thecorresponding position from SEQ ID NO:2.
 20. The isolated polynucleotideof claim 10, further comprising the nucleotide sequence set forth in SEQID NO:23.
 21. The isolated polynucleotide of claim 20, wherein thehumanized heavy chain variable region is encoded by the nucleotidesequence set forth in SEQ ID NO:13 or SEQ ID NO:17, and the humanizedlight chain variable region is encoded by the sequence set forth in SEQID NO:23.
 22. A vector comprising the isolated polynucleotide ofclaim
 1. 23. A vector comprising the isolated polynucleotide of claim11.
 24. A host cell transfected with the vector of claim
 22. 25. A hostcell transfected with the vector of claim
 23. 26. The host cell of claim24 that is a mammalian host cell.
 27. The host cell of claim 26, whereinthe mammalian host cell is selected from the group consisting of CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138 cells.
 28. The host cellof claim 25 that is a mammalian host cell.
 29. The host cell of claim28, wherein the mammalian host cell is selected from the groupconsisting of CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138 cells.30. A method of producing an antibody, comprising culturing the hostcell of claim 24 to produce the antibody.
 31. A method of producing anantibody, comprising culturing the host cell of claim 25 to produce theantibody.